Amplification of denatured and stabilized nucleic acids

ABSTRACT

Disclosed are compositions and a method for amplification of nucleic acid sequences of interest. The disclosed method generally involves replication of a target sequence such that, during replication, the replicated strands are displaced from the target sequence by strand displacement replication of another replicated strand. In one form of the disclosed method, the target sample is not subjected to denaturing conditions. It was discovered that the target nucleic acids, genomic DNA, for example, need not be denatured for efficient multiple displacement amplification. The primers used can be hexamer primers. The primers can also each contain at least one modified nucleotide such that the primers are nuclease resistant. The primers can also each contain at least one modified nucleotide such that the melting temperature of the primer is altered relative to a primer of the same sequence without the modified nucleotide(s). The DNA polymerase can be φ29 DNA polymerase.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.09/982,212, filed Oct. 18, 2001, now U.S. Pat. No. 6,617,137, which is acontinuation of copending application ser. No. 09/977,868, filed Oct.15, 2001 now U.S. Pat. No. 6,977,148, both of which are herebyincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The disclosed invention is generally in the field of nucleic acidamplification.

BACKGROUND OF THE INVENTION

A number of methods have been developed for exponential amplification ofnucleic acids. These include the polymerase chain reaction (PCR), ligasechain reaction (LCR), self-sustained sequence replication (3SR), nucleicacid sequence based amplification (NASBA), strand displacementamplification (SDA), and amplification with Qβ replicase (Birkenmeyerand Mushahwar, J. Virological Methods, 35:117–126 (1991); Landegren,Trends Genetics 9:199–202 (1993)).

Fundamental to most genetic analysis is availability of genomic DNA ofadequate quality and quantity. Since DNA yield from human samples isfrequently limiting, much effort has been invested in general methodsfor propagating and archiving genomic DNA. Methods include the creationof EBV-transformed cell lines or whole genome amplification (WGA) byrandom or degenerate oligonucleotide-primed PCR. Whole genome PCR, avariant of PCR amplification, involves the use of random or partiallyrandom primers to amplify the entire genome of an organism in the samePCR reaction. This technique relies on having a sufficient number ofprimers of random or partially random sequence such that pairs ofprimers will hybridize throughout the genomic DNA at moderate intervals.Replication initiated at the primers can then result in replicatedstrands overlapping sites where another primer can hybridize. Bysubjecting the genomic sample to multiple amplification cycles, thegenomic sequences will be amplified. Whole genome PCR has the samedisadvantages as other forms of PCR. However, WGA methods suffer fromhigh cost or insufficient coverage and inadequate average DNA size(Telenius et al., Genomics. 13:718–725 (1992); Cheung and Nelson, ProcNatl Acad Sci USA. 93:14676–14679 (1996); Zhang et al., Proc Natl AcadSci USA. 89:5847–5851 (1992)).

Another form of nucleic acid amplification, involving stranddisplacement, has been described in U.S. Pat. No. 6,124,120 to Lizardi.In one form of the method, two sets of primers are used that arecomplementary to opposite strands of nucleotide sequences flanking atarget sequence. Amplification proceeds by replication initiated at eachprimer and continuing through the target nucleic acid sequence, with thegrowing strands encountering and displacing previously replicatedstrands. In another form of the method a random set of primers is usedto randomly prime a sample of genomic nucleic acid. The primers in theset are collectively, and randomly, complementary to nucleic acidsequences distributed throughout nucleic acid in the sample.Amplification proceeds by replication initiating at each primer andcontinuing so that the growing strands encounter and displace adjacentreplicated strands. In another form of the method concatenated DNA isamplified by strand displacement synthesis with either a random set ofprimers or primers complementary to linker sequences between theconcatenated DNA. Synthesis proceeds from the linkers, through a sectionof the concatenated DNA to the next linker, and continues beyond, withthe growing strands encountering and displacing previously replicatedstrands.

BRIEF SUMMARY OF THE INVENTION

Disclosed are compositions and a method for amplification of nucleicacid sequences of interest. The method is based on strand displacementreplication of the nucleic acid sequences by multiple primers. Thedisclosed method, referred to as multiple displacement amplification(MDA), improves on prior methods of strand displacement replication. Thedisclosed method generally involves bringing into contact a set ofprimers, DNA polymerase, and a target sample, and incubating the targetsample under conditions that promote replication of the target sequence.Replication of the target sequence results in replicated strands suchthat, during replication, the replicated strands are displaced from thetarget sequence by strand displacement replication of another replicatedstrand.

In some forms of the disclosed method, a genomic sample is prepared byexposing cells to alkaline conditions, thereby lysing the cells andresulting in a cell lysate; reducing the pH of the cell lysate to makethe pH of the cell lysate compatible with DNA replication; andincubating the cell lysate under conditions that promote replication ofthe genome of the cells by multiple displacement amplification. It hasbeen discovered that alkaline lysis can cause less damage to genomic DNAand that alkaline lysis is compatible with multiple displacementamplification. The alkaline conditions can be, for example, those thatcause a substantial number of cells to lyse or those that cause asufficient number of cells to lyse. The number of lysed cells can beconsidered sufficient if the genome can be sufficiently amplified in thedisclosed method. The amplification is sufficient if enoughamplification product is produced to permit some use of theamplification product, such as detection of sequences or other analysis.The reduction in pH is generally into the neutral range of pH 9.0 to pH6.0.

In some embodiments, the cells are not lysed by heat and/or the nucleicacids in the cell lysate is not denatured by heating. Those of skill inthe art will understand that different cells under different conditionswill be lysed at different temperatures and so can determinetemperatures and times at which the cells will not be lysed by heat. Ingeneral, the cells are not subjected to heating above a temperature andfor a time that would cause substantial cell lysis in the absence of thealkaline conditions used. In some embodiments, the cells and/or celllysate are not subjected to heating substantially above the temperatureat which the cells grow. In other embodiments, the cells and/or celllysate are not subjected to heating substantially above the temperatureof the amplification reaction (where the genome is replicated). Thedisclosed multiple displacement amplification reaction is generallyconducted at a substantially constant temperature (that is, theamplification reaction is substantially isothermic), and thistemperature is generally below the temperature at which the nucleicacids would be substantially or significantly denatured.

In some embodiments, the cell lysate is not subjected to purificationprior to the amplification reaction. In the context of the disclosedmethod, purification generally refers to the separation of nucleic acidsfrom other material in the cell lysate. It has been discovered thatmultiple displacement amplification can be performed on unpurified andpartially purified samples. It is commonly thought that amplificationreactions cannot be efficiently performed using unpurified nucleic acid.In particular, PCR is very sensitive to contaminants.

In some forms of the disclosed method, the target sample is notsubjected to denaturing conditions. It was discovered that the targetnucleic acids, genomic DNA, for example, need not be denatured forefficient multiple displacement amplification. It was discovered thatelimination of a denaturation step and denaturation conditions hasadditional advantages such as reducing sequence bias in the amplifiedproducts. In another embodiment, the primers can be hexamer primers. Itwas discovered that such short, 6 nucleotide primers can still primemultiple strand displacement replication efficiently. Such short primersare easier to produce as a complete set of primers of random sequence(random primers) than longer primers because there are fewer separatespecies of primers in a pool of shorter primers. In another embodiment,the primers can each contain at least one modified nucleotide such thatthe primers are nuclease resistant. In another embodiment, the primerscan each contain at least one modified nucleotide such that the meltingtemperature of the primer is altered relative to a primer of the samesequence without the modified nucleotide(s). For these last twoembodiments, it is preferred that the primers are modified RNA. Inanother embodiment, the DNA polymerase can be φ29 DNA polymerase. It wasdiscovered that φ29 DNA polymerase produces greater amplification inmultiple displacement amplification. The combination of two or more ofthe above features also yields improved results in multiple displacementamplification. In a preferred embodiment, for example, the target sampleis not subjected to denaturing conditions, the primers are hexamerprimers and contain modified nucleotides such that the primers arenuclease resistant, and the DNA polymerase is φ29 DNA polymerase. Theabove features are especially useful in whole genome strand displacementamplification (WGSDA).

In some forms of the disclosed method, the method includes labeling ofthe replicated strands (that is, the strands produced in multipledisplacement amplification) using terminal deoxynucleotidyl transferase.The replicated strands can be labeled by, for example, the addition ofmodified nucleotides, such as biotinylated nucleotides, fluorescentnucleotides, 5 methyl dCTP, bromodeoxyuridine triphosphate (BrdUTP), or5-(3-aminoallyl)-2′-deoxyuridine 5′-triphosphates, to the 3′ ends of thereplicated strands. The replicated strands can also be labeled byincorporating modified nucleotides during replication. Probes replicatedin this manner are particularly useful for hybridization, including usein microarray formats.

In one form of the disclosed method, referred to as whole genome stranddisplacement amplification (WGSDA), a random set of primers is used torandomly prime a sample of genomic nucleic acid (or another sample ofnucleic acid of high complexity). By choosing a sufficiently large setof primers of random or partially random sequence, the primers in theset will be collectively, and randomly, complementary to nucleic acidsequences distributed throughout nucleic acid in the sample.Amplification proceeds by replication with a highly processivepolymerase initiating at each primer and continuing until spontaneoustermination. A key feature of this method is the displacement ofintervening primers during replication by the polymerase. In this way,multiple overlapping copies of the entire genome can be synthesized in ashort time. The method has advantages over the polymerase chain reactionsince it can be carried out under isothermal conditions. Otheradvantages of whole genome strand displacement amplification include ahigher level of amplification than whole genome PCR (up to five timeshigher), amplification is less sequence-dependent than PCR, and thereare no re-annealing artifacts or gene shuffling artifacts as can occurwith PCR (since there are no cycles of denaturation and re-annealing).In preferred embodiments of WGSDA, the target sample is not subjected todenaturing conditions, the primers are hexamer primers and containmodified nucleotides such that the primers are nuclease resistant, theDNA polymerase is φ29 DNA polymerase, or any combination of thesefeatures.

In another form of the method, referred to as multiple stranddisplacement amplification (MSDA), two sets of primers are used, a rightset and a left set. Primers in the right set of primers each have aportion complementary to nucleotide sequences flanking one side of atarget nucleotide sequence and primers in the left set of primers eachhave a portion complementary to nucleotide sequences flanking the otherside of the target nucleotide sequence. The primers in the right set arecomplementary to one strand of the nucleic acid molecule containing thetarget nucleotide sequence and the primers in the left set arecomplementary to the opposite strand. The 5′ end of primers in both setsare distal to the nucleic acid sequence of interest when the primers arehybridized to the flanking sequences in the nucleic acid molecule.Preferably, each member of each set has a portion complementary to aseparate and non-overlapping nucleotide sequence flanking the targetnucleotide sequence. Amplification proceeds by replication initiated ateach primer and continuing through the target nucleic acid sequence. Inanother form of MSDA, referred to as linear MSDA, amplification isperformed with a set of primers complementary to only one strand, thusamplifying only one of the strands.

In another form of the method, referred to as gene specific stranddisplacement amplification (GS-MSDA), target DNA is first digested witha restriction endonuclease. The digested fragments are then ligatedend-to-end to form DNA circles. These circles can be monomers orconcatemers. Two sets of primers are used for amplification, a right setand a left set. Primers in the right set of primers each have a portioncomplementary to nucleotide sequences flanking one side of a targetnucleotide sequence and primers in the left set of primers each have aportion complementary to nucleotide sequences flanking the other side ofthe target nucleotide sequence. The primers in the right set arecomplementary to one strand of the nucleic acid molecule containing thetarget nucleotide sequence and the primers in the left set arecomplementary to the opposite strand. The primers are designed to coverall or part of the sequence needed to be amplified. Preferably, eachmember of each set has a portion complementary to a separate andnon-overlapping nucleotide sequence flanking the target nucleotidesequence. Amplification proceeds by replication initiated at each primerand continuing through the target nucleic acid sequence. In one form ofGS-MSDA, referred to as linear GS-MSDA, amplification is performed witha set of primers complementary to only one strand, thus amplifying onlyone of the strands. In another form of GS-MSDA, cDNA sequences can becircularized to form single stranded DNA circles. Amplification is thenperformed with a set of primers complementary to the single-strandedcircular cDNA.

A key feature of this method is the displacement of intervening primersduring replication. Once the nucleic acid strands elongated from theright set of primers reaches the region of the nucleic acid molecule towhich the left set of primers hybridizes, and vice versa, another roundof priming and replication will take place. This allows multiple copiesof a nested set of the target nucleic acid sequence to be synthesized ina short period of time. By using a sufficient number of primers in theright and left sets, only a few rounds of replication are required toproduce hundreds of thousands of copies of the nucleic acid sequence ofinterest. The disclosed method has advantages over the polymerase chainreaction since it can be carried out under isothermal conditions. Nothermal cycling is needed because the polymerase at the head of anelongating strand (or a compatible strand-displacement protein) willdisplace, and thereby make available for hybridization, the strand aheadof it. Other advantages of multiple strand displacement amplificationinclude the ability to amplify very long nucleic acid segments (on theorder of 50 kilobases) and rapid amplification of shorter segments (10kilobases or less). In multiple strand displacement amplification,single priming events at unintended sites will not lead to artifactualamplification at these sites (since amplification at the intended sitewill quickly outstrip the single strand replication at the unintendedsite). In preferred embodiments of MSDA, the target sample is notsubjected to denaturing conditions, the primers are hexamer primers andcontain modified nucleotides such that the primers are nucleaseresistant, the DNA polymerase is φ29 DNA polymerase, or any combinationof these features.

In preferred embodiments of WGSDA, the target sample is not subjected todenaturing conditions, the primers are hexamer primers and containmodified nucleotides such that the primers are nuclease resistant, theDNA polymerase is φ29 DNA polymerase, or any combination of thesefeatures.

Following amplification, the amplified sequences can be used for anypurpose, such as uses known and established for PCR amplified sequences.For example, amplified sequences can be detected using any of theconventional detection systems for nucleic acids such as detection offluorescent labels, enzyme-linked detection systems, antibody-mediatedlabel detection, and detection of radioactive labels. A preferred formof labeling involves labeling of the replicated strands (that is, thestrands produced in multiple displacement amplification) using terminaldeoxynucleotidyl transferase. The replicated strands can be labeled by,for example, the addition of modified nucleotides, such as biotinylatednucleotides, fluorescent nucleotides, 5 methyl dCTP, BrdUTP, or5-(3-aminoallyl)-2′-deoxyuridine 5′-triphosphates, to the 3′ ends of thereplicated strands.

In the disclosed method amplification takes place not in cycles, but ina continuous, isothermal replication. This makes amplification lesscomplicated and much more consistent in output. Strand displacementallows rapid generation of multiple copies of a nucleic acid sequence orsample in a single, continuous, isothermal reaction. DNA that has beenproduced using the disclosed method can then be used for any purpose orin any other method desired. For example, PCR can be used to furtheramplify any specific DNA sequence that has been previously amplified bythe whole genome strand displacement method.

Genetic analysis must frequently be carried out with DNA derived frombiological samples, such as blood, tissue culture cells, buccal swabs,mouthwash, stool, tissues slices, biopsy aspiration, and archeologicalsamples such as bone or mummified tissue. In some cases, the samples aretoo small to extract a sufficient amount of pure DNA and it is necessaryto carry out DNA-based assays directly from the unprocessed sample.Furthermore, it is time consuming to isolate pure DNA, and so thedisclosed method, which can amplify the genome directly from biologicalsamples, represents a substantial improvement.

The disclosed method has several distinct advantages over currentmethodologies. The genome can be amplified directly from whole blood orcultured cells with simple cell lysis techniques such as KOH treatment.PCR and other DNA amplification methods are severely inhibited bycellular contents and so purification of DNA is needed prior toamplification and assay. For example, heme present in lysed blood cellsinhibits PCR. In contrast, the disclosed form of whole genomeamplification can be carried out on crude lysates with no need tophysically separate DNA by miniprep extraction and precipitationprocedures, or with column or spin cartridge methods.

Bacteria, fungi, and viruses may all be involved in nosocomialinfections. Identification of nosocomial pathogens at the sub-specieslevel requires sophisticated discriminatory techniques. Such techniquesutilize traditional as well as molecular methods for typing. Sometraditional techniques are antimicrobial susceptibility testing,determination of the ability to utilize biochemical substrates, andserotyping. A major limitation of these techniques is that they takeseveral days to complete, since they require pure bacterial cultures.Because such techniques are long, and the bacteria may even benon-viable in the clinical samples, there is a need to have a quick andreliable method for bacterial species identification.

Some of the DNA-based molecular methods for the identification ofbacterial species are macrorestriction analysis (MRA) followed bypulsed-field gel electrophoresis (PFGE), amplified fragment lengthpolymorphism (AFLP) analysis, and arbitrarily primed PCR (AP-PCR)(Tenover et al., J. Clin. Microbiol. 32:407–415 (1994), and Pruckler etal., J. Clin. Microbiol. 33:2872–2875 (1995)). These moleculartechniques are labor-intensive and difficult to standardize amongdifferent laboratories.

The disclosed method provides a useful alternative method for theidentification of bacterial strains by amplification of microbial DNAfor analysis. Unlike PCR (Lantz et al., Biotechnol. Annu. Rev. 5:87–130(2000)), the disclosed method is rapid, non-biased, reproducible, andcapable of amplifying large DNA segments from bacterial, viral or fungalgenomes.

The disclosed method can be used, for example, to obtain enough DNA fromunculturable organisms for sequencing or other studies. Mostmicroorganisms cannot be propagated outside their native environment,and therefore their nucleic acids cannot be sequenced. Many unculturableorganisms live under extreme conditions, which makes their geneticcomplement of interest to investigators. Other microorganisms live incommunities that play a vital role in certain ecosystems. Individualorganisms or entire communities of organisms can be amplified andsequenced, individually or together.

Recombinant proteins may be purified from a large biomass grown up frombacterial or yeast strains harboring desired expression vectors. A highdegree of purity may be desired for the isolated recombinant protein,requiring a sensitive procedure for the detection of trace levels ofprotein or DNA contaminants. The disclosed method is a DNA amplificationreaction that is highly robust even in the presence of low levels of DNAtemplate, and can be used to monitor preparations of recombinant proteinfor trace amounts of contaminating bacterial or yeast genomic DNA.

Amplification of forensic material for RFLP-based testing is one usefulapplication for the disclosed method.

Also disclosed is a method for amplifying and repairing damaged DNA.This method is useful, for example, for amplifying degraded genomic DNA.The method involves substantially denaturing a damaged DNA sample(generally via exposure to heat and alkaline conditions), removal orreduction of the denaturing conditions (such as by reduction of the pHand temperature of the denatured DNA sample), and replicating the DNA.The damaged DNA is repaired during replication by increasing the averagelength of the damaged DNA. For example, the average length of DNAfragments can be increase from, for example, 2 kb in the damaged DNAsample to, for example, 10 kb or greater for the replicated DNA. Thisrepair method can result in an overall improvement in amplification ofdamaged DNA by increasing the average length of the product, increasingthe quality of the amplification products by 3-fold (by, for example,increasing the marker representation in the sample), and improving thegenotyping of amplified products by lowering the frequency of allelicdropout; all compared to the results when amplifying damaged DNA byother methods. The removal of denaturing conditions can allow denaturedstrands of damaged DNA to hybridize to other denatured damaged DNA. Thereplication can be multiple displacement amplification. Substantialdenaturation and transient denaturation of the DNA samples generally iscarried out such that the DNA is not further damaged. This method cangenerally be combined or used with any of the disclosed amplificationmethods.

It is an object of the disclosed invention to provide a method ofamplifying a target nucleic acid sequence in a continuous, isothermalreaction.

It is another object of the disclosed invention to provide a method ofamplifying an entire genome or other highly complex nucleic acid samplein a continuous, isothermal reaction.

It is another object of the disclosed invention to provide a method ofamplifying a target nucleic acid sequence where multiple copies of thetarget nucleic acid sequence are produced in a single amplificationcycle.

It is another object of the disclosed invention to provide a method ofamplifying a concatenated DNA in a continuous, isothermal reaction.

It is another object of the disclosed invention to provide a kit foramplifying a target nucleic acid sequence in a continuous, isothermalreaction.

It is another object of the disclosed invention to provide a kit foramplifying an entire genome or other highly complex nucleic acid samplein a continuous, isothermal reaction.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of DNA synthesis (in μg) versus time (in hours) usingdifferent amounts of nucleic acid for amplification in the disclosedmethod.

FIG. 2 is a graph of the effect of incubation time at 95° C. on templateDNA length.

FIG. 3 is a graph of the effect of template incubation at 95° C. on therate and yield of MDA.

FIG. 4 is a graph of the effect of template incubation at 95° C. on theaverage size of DNA product strands.

FIG. 5 is a graph showing a comparison of the effect of templateincubation at 95° C. versus no incubation at 95° C. on locusrepresentation in DNA amplified by MDA.

FIGS. 6A, 6B, and 6C are graphs showing the effect of amplification ongene representation bias for three different amplification procedures,MDA, DOP-PCR, and PEP.

FIG. 7 is a graph showing amplification of c-jun sequences using nestedprimers.

FIG. 8 is a graph the relative representation of eight loci for DNA fromfive different amplification reactions. The Y-axis is the locusrepresentation, expressed as a percent, relative to input genomic DNA,which is calculated as the yield of quantitative PCR product from 1 μgof amplified DNA divided by the yield from 1 μg of genomic DNA control.

FIG. 9 is a graph showing a comparison of the percent representation for8 loci for DNA amplified in a reaction containing 100% dTTP and DNAamplified in a reaction containing 30% dTTP/70% AAdUTP.

FIG. 10 is a graph showing the amplification of c-jun sequences usingcircularized genomic template. The Y-axis is the locus representation,expressed as a percent, relative to input genomic DNA, which iscalculated as the yield of quantitative PCR product from 1 μg ofamplified DNA divided by the yield from 1 μg of genomic DNA control.

FIG. 11 is a graph showing a comparison of the percent representationfor 8 loci in DNA amplified using c-jun specific primers andcircularized DNA target.

FIG. 12 is a graph of percent locus representation of different DNAsamples exposed to different treatments (control or repair treatments).

FIG. 13 is a graph of percent locus representation of 40 samples with orwithout repair treatment.

DETAILED DESCRIPTION OF THE INVENTION

The disclosed method makes use of certain materials and procedures whichallow amplification of target nucleic acid sequences and whole genomesor other highly complex nucleic acid samples. These materials andprocedures are described in detail below.

Materials

A. Target Sequence

The target sequence, which is the object of amplification, can be anynucleic acid. The target sequence can include multiple nucleic acidmolecules, such as in the case of whole genome amplification, multiplesites in a nucleic acid molecule, or a single region of a nucleic acidmolecule. For multiple strand displacement amplification, generally thetarget sequence is a single region in a nucleic acid molecule or nucleicacid sample. For whole genome amplification, the target sequence is theentire genome or nucleic acid sample. A target sequence can be in anynucleic acid sample of interest. The source, identity, and preparationof many such nucleic acid samples are known. It is preferred thatnucleic acid samples known or identified for use in amplification ordetection methods be used for the method described herein. The nucleicacid sample can be, for example, a nucleic acid sample from one or morecells, tissue, or bodily fluids such as blood, urine, semen, lymphaticfluid, cerebrospinal fluid, or amniotic fluid, or other biologicalsamples, such as tissue culture cells, buccal swabs, mouthwash, stool,tissues slices, biopsy aspiration, and archeological samples such asbone or mummified tissue. Types of useful target samples include bloodsamples, urine samples, semen samples, lymphatic fluid samples,cerebrospinal fluid samples, amniotic fluid samples, biopsy samples,needle aspiration biopsy samples, cancer samples, tumor samples, tissuesamples, cell samples, cell lysate samples, a crude cell lysate samples,forensic samples, archeological samples, infection samples, nosocomialinfection samples, production samples, drug preparation samples,biological molecule production samples, protein preparation samples,lipid preparation samples, and/or carbohydrate preparation samples.

For multiple strand displacement amplification, preferred targetsequences are those which are difficult to amplify using PCR due to, forexample, length or composition. For whole genome amplification,preferred target sequences are nucleic acid samples from a single cell.For multiple strand displacement amplification of concatenated DNA thetarget is the concatenated DNA. The target sequence can be either one orboth strands of cDNA. The target sequences for use in the disclosedmethod are preferably part of nucleic acid molecules or samples that arecomplex and non-repetitive (with the exception of the linkers inlinker-concatenated DNA and sections of repetitive DNA in genomic DNA).

Target nucleic acids can include damaged DNA and damaged DNA samples.For example, preparation of genomic DNA samples can result in damage tothe genomic DNA (for example, degradation and fragmentation). This canmake amplification of the genome or sequences in it both more difficultand provide less reliable results (by, for example, resulting inamplification of many partial and fragmented genomic sequences. DamagedDNA and damaged DNA samples are thus useful for the disclosed method ofamplifying damaged DNA. Any degraded, fragmented or otherwise damagedDNA or sample containing such DNA can be used in the disclosed method.

1. Target Sequences for Multiple Strand Displacement Amplification

Although multiple sites in a nucleic acid sample can be amplifiedsimultaneously in the same MSDA reaction, for simplicity, the followingdiscussion will refer to the features of a single nucleic acid sequenceof interest which is to be amplified. This sequence is referred to belowas a target sequence. It is preferred that a target sequence for MSDAinclude two types of target regions, an amplification target and ahybridization target. The hybridization target includes the sequences inthe target sequence that are complementary to the primers in a set ofprimers. The amplification target is the portion of the target sequencewhich is to be amplified. For this purpose, the amplification target ispreferably downstream of, or flanked by the hybridization target(s).There are no specific sequence or structural requirements for choosing atarget sequence. The hybridization target and the amplification targetwithin the target sequence are defined in terms of the relationship ofthe target sequence to the primers in a set of primers. The primers aredesigned to match the chosen target sequence. Although preferred, it isnot required that sequences to be amplified and the sites ofhybridization of the primers be separate since sequences in and aroundthe sites where the primers hybridize will be amplified.

In multiple strand displacement amplification of circularized DNA, thecircular DNA fragments are the amplification targets. The hybridizationtargets include the sequences that are complementary to the primers usedfor amplification. One form of circular DNA for amplification iscircularized cDNA.

In multiple strand displacement amplification of linker-concatenatedDNA, the DNA fragments joined by the linkers are the amplificationtargets and the linkers are the hybridization target. The hybridizationtargets (that is, the linkers) include the sequences that arecomplementary to the primers used for amplification. One form ofconcatenated DNA for amplification is concatenated cDNA.

B. Primers

Primers for use in the disclosed amplification method areoligonucleotides having sequence complementary to the target sequence.This sequence is referred to as the complementary portion of the primer.The complementary portion of a primer can be any length that supportsspecific and stable hybridization between the primer and the targetsequence under the reaction conditions. Generally, for reactions at 37°C., this can be 10 to 35 nucleotides long or 16 to 24 nucleotides long.For whole genome amplification, the primers can be from 5 to 60nucleotides long, and in particular, can be 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, and/or 20 nucleotides long.

For some forms of the disclosed method, such as those using primers orrandom or degenerate sequence (that is, use of a collection of primershaving a variety of sequences), primer hybridization need not bespecific. In such cases the primers need only be effective in primingsynthesis. For example, in whole genome amplification specificity ofpriming is not essential since the goal generally is to amplify allsequences equally. Sets of random or degenerate primers can be composedof primers 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and/or20 nucleotides long or more. Primers six nucleotides long are referredto as hexamer primers. Preferred primers for whole genome amplificationare random hexamer primers, for example, random hexamer primers whereevery possible six nucleotide sequence is represented in the set ofprimers. Similarly, sets of random primers of other particular lengths,or of a mixture of lengths preferably contain every possible sequencethe length of the primer, or, in particular, the length of thecomplementary portion of the primer. Use of random primers is describedin U.S. Pat. Nos. 5,043,272 and 6,214,587.

The disclosed primers can have one or more modified nucleotides. Suchprimers are referred to herein as modified primers. Modified primershave several advantages. First, some forms of modified primers, such asRNA/2′-O-methyl RNA chimeric primers, have a higher melting temperature(Tm) than DNA primers. This increases the stability of primerhybridization and will increase strand invasion by the primers. Thiswill lead to more efficient priming. Also, since the primers are made ofRNA, they will be exonuclease resistant. Such primers, if tagged withminor groove binders at their 5′ end, will also have better strandinvasion of the template dsDNA. In addition, RNA primers can also bevery useful for WGA from biological samples such as cells or tissue.Since the biological samples contain endogenous RNA, this RNA can bedegraded with RNase to generate a pool of random oligomers, which canthen be used to prime the polymerase for amplification of the DNA. Thiseliminates any need to add primers to the reaction. Alternatively, DNasedigestion of biological samples can generate a pool of DNA oligo primersfor RNA dependent DNA amplification.

Chimeric primers can also be used. Chimeric primers are primers havingat least two types of nucleotides, such as both deoxyribonucleotides andribonucleotides, ribonucleotides and modified nucleotides, or twodifferent types of modified nucleotides. One form of chimeric primer ispeptide nucleic acid/nucleic acid primers. For example, 5′-PNA-DNA-3′ or5′-PNA-RNA-3′ primers may be used for more efficient strand invasion andpolymerization invasion. The DNA and RNA portions of such primers canhave random or degenerate sequences. Other forms of chimeric primersare, for example, 5′-(2′-O-Methyl) RNA-RNA-3′ or 5′-(2′-O-Methyl)RNA-DNA-3′.

Many modified nucleotides (nucleotide analogs) are known and can be usedin oligonucleotides. A nucleotide analog is a nucleotide which containssome type of modification to either the base, sugar, or phosphatemoieties. Modifications to the base moiety would include natural andsynthetic modifications of A, C, G, and T/U as well as different purineor pyrimidine bases, such as uracil-5-yl, hypoxanthin-9-yl (I), and2-aminoadenin-9-yl. A modified base includes but is not limited to5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives ofadenine and guanine, 2-propyl and other alkyl derivatives of adenine andguanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouraciland cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine andthymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Additional basemodifications can be found for example in U.S. Pat. No. 3,687,808,Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and Sanghvi, Y. S., Chapter 15, Antisense Research andApplications, pages 289–302, Crooke, S. T. and Lebleu, B. ed., CRCPress, 1993. Certain nucleotide analogs, such as 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine can increase the stability of duplex formation. Othermodified bases are those that function as universal bases. Universalbases include 3-nitropyrrole and 5-nitroindole. Universal basessubstitute for the normal bases but have no bias in base pairing. Thatis, universal bases can base pair with any other base. Primers composed,either in whole or in part, of nucleotides with universal bases areuseful for reducing or eliminating amplification bias against repeatedsequences in a target sample. This would be useful, for example, where aloss of sequence complexity in the amplified products is undesirable.Base modifications often can be combined with for example a sugarmodification, such as 2′-O-methoxyethyl, to achieve unique propertiessuch as increased duplex stability. There are numerous United Statespatents such as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066;5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908;5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091;5,614,617; and 5,681,941, which detail and describe a range of basemodifications. Each of these patents is herein incorporated byreference.

Nucleotide analogs can also include modifications of the sugar moiety.Modifications to the sugar moiety would include natural modifications ofthe ribose and deoxyribose as well as synthetic modifications. Sugarmodifications include but are not limited to the following modificationsat the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-,S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl andalkynyl may be substituted or unsubstituted C1 to C10, alkyl or C2 toC10 alkenyl and alkynyl. 2′ sugar modifications also include but are notlimited to —O[(CH₂)nO]mCH₃, —O(CH₂)nOCH₃, —O(CH₂)nNH₂, —O(CH₂)nCH₃,—O(CH₂)n—ONH2, and —O(CH₂)nON[(CH₂)nCH₃)]₂, where n and m are from 1 toabout 10.

Other modifications at the 2′ position include but are not limited to:C1 to C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl,O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, a group for improving thepharmacokinetic properties of an oligonucleotide, or a group forimproving the pharmacodynamic properties of an oligonucleotide, andother substituents having similar properties. Similar modifications mayalso be made at other positions on the sugar, particularly the 3′position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide. Modifiedsugars would also include those that contain modifications at thebridging ring oxygen, such as CH₂ and S. Nucleotide sugar analogs mayalso have sugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. There are numerous United States patents thatteach the preparation of such modified sugar structures such as U.S.Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265;5,658,873; 5,670,633; and 5,700,920, each of which is hereinincorporated by reference in its entirety.

Nucleotide analogs can also be modified at the phosphate moiety.Modified phosphate moieties include but are not limited to those thatcan be modified so that the linkage between two nucleotides contains aphosphorothioate, chiral phosphorothioate, phosphorodithioate,phosphotriester, aminoalkylphosphotriester, methyl and other alkylphosphonates including 3′-alkylene phosphonate and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates. It is understood that these phosphate or modifiedphosphate linkages between two nucleotides can be through a 3′-5′linkage or a 2′-5′ linkage, and the linkage can contain invertedpolarity such as 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixedsalts and free acid forms are also included. Numerous United Statespatents teach how to make and use nucleotides containing modifiedphosphates and include but are not limited to, U.S. Pat. Nos. 3,687,808;4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423;5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939;5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821;5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050,each of which is herein incorporated by reference.

It is understood that nucleotide analogs need only contain a singlemodification, but may also contain multiple modifications within one ofthe moieties or between different moieties.

Nucleotide substitutes are molecules having similar functionalproperties to nucleotides, but which do not contain a phosphate moiety,such as peptide nucleic acid (PNA). Nucleotide substitutes are moleculesthat will recognize and hybridize to complementary nucleic acids in aWatson-Crick or Hoogsteen manner, but which are linked together througha moiety other than a phosphate moiety. Nucleotide substitutes are ableto conform to a double helix type structure when interacting with theappropriate target nucleic acid.

Nucleotide substitutes are nucleotides or nucleotide analogs that havehad the phosphate moiety and/or sugar moieties replaced. Nucleotidesubstitutes do not contain a standard phosphorus atom. Substitutes forthe phosphate can be for example, short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatom and alkyl or cycloalkylinternucleoside linkages, or one or more short chain heteroatomic orheterocyclic internucleoside linkages. These include those havingmorpholino linkages (formed in part from the sugar portion of anucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH2 component parts. Numerous United States patents disclosehow to make and use these types of phosphate replacements and includebut are not limited to U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444;5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938;5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225;5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289;5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439,each of which is herein incorporated by reference.

It is also understood in a nucleotide substitute that both the sugar andthe phosphate moieties of the nucleotide can be replaced, by for examplean amide type linkage (aminoethylglycine) (PNA). U.S. Pat. Nos.5,539,082; 5,714,331; and 5,719,262 teach how to make and use PNAmolecules, each of which is herein incorporated by reference. (See alsoNielsen et al., Science 254:1497–1500 (1991)).

Primers can be comprised of nucleotides and can be made up of differenttypes of nucleotides or the same type of nucleotides. For example, oneor more of the nucleotides in a primer can be ribonucleotides,2′-O-methyl ribonucleotides, or a mixture of ribonucleotides and2′-O-methyl ribonucleotides; about 10% to about 50% of the nucleotidescan be ribonucleotides, 2′-O-methyl ribonucleotides, or a mixture ofribonucleotides and 2′-O-methyl ribonucleotides; about 50% or more ofthe nucleotides can be ribonucleotides, 2′-O-methyl ribonucleotides, ora mixture of ribonucleotides and 2′-O-methyl ribonucleotides; or all ofthe nucleotides are ribonucleotides, 2′-O-methyl ribonucleotides, or amixture of ribonucleotides and 2′-O-methyl ribonucleotides. Thenucleotides can be comprised of bases (that is, the base portion of thenucleotide) and can (and normally will) comprise different types ofbases. For example, one or more of the bases can be universal bases,such as 3-nitropyrrole or 5-nitroindole; about 10% to about 50% of thebases can be universal bases; about 50% or more of the bases can beuniversal bases; or all of the bases can be universal bases.

Primers may, but need not, also contain additional sequence at the 5′end of the primer that is not complementary to the target sequence. Thissequence is referred to as the non-complementary portion of the primer.The non-complementary portion of the primer, if present, serves tofacilitate strand displacement during DNA replication. Thenon-complementary portion of the primer can also include a functionalsequence such as a promoter for an RNA polymerase. The non-complementaryportion of a primer may be any length, but is generally 1 to 100nucleotides long, and preferably 4 to 8 nucleotides long. The use of anon-complementary portion is not preferred when random or partiallyrandom primers are used for whole genome amplification.

1. Primers for Whole Genome Strand Displacement Amplification

In the case of whole genome strand displacement amplification, it ispreferred that a set of primers having random or partially randomnucleotide sequences be used. In a nucleic acid sample of significant orsubstantial complexity, which is the preferred target sequence forWGSDA, specific nucleic acid sequences present in the sample need not beknown and the primers need not be designed to be complementary to anyparticular sequence. Rather, the complexity of the nucleic acid sampleresults in a large number of different hybridization target sequences inthe sample which will be complementary to various primers of random orpartially random sequence. The complementary portion of primers for usein WGSDA can be fully randomized, have only a portion that israndomized, or be otherwise selectively randomized.

The number of random base positions in the complementary portion ofprimers are preferably from 20% to 100% of the total number ofnucleotides in the complementary portion of the primers. More preferablythe number of random base positions are from 30% to 100% of the totalnumber of nucleotides in the complementary portion of the primers. Mostpreferably the number of random base positions are from 50% to 100% ofthe total number of nucleotides in the complementary portion of theprimers. Sets of primers having random or partially random sequences canbe synthesized using standard techniques by allowing the addition of anynucleotide at each position to be randomized. It is also preferred thatthe sets of primers are composed of primers of similar length and/orhybridization characteristics.

2. Primers for Multiple Strand Displacement Amplification

In the case of multiple strand displacement amplification, thecomplementary portion of each primer is designed to be complementary tothe hybridization target in the target sequence. In a set of primers, itis preferred that the complementary portion of each primer becomplementary to a different portion of the target sequence. It is morepreferred that the primers in the set be complementary to adjacent sitesin the target sequence. It is also preferred that such adjacent sites inthe target sequence are also adjacent to the amplification target in thetarget sequence.

It is preferred that, when hybridized to a target sequence, the primersin a set of primers are separated from each other. It is preferred that,when hybridized, the primers in a set of primers are separated from eachother by at least 5 bases. It is more preferred that, when hybridized,the primers in a set of primers are separated from each other by atleast 10 bases. It is still more preferred that, when hybridized, theprimers in a set of primers are separated from each other by at least 20bases. It is still more preferred that, when hybridized, the primers ina set of primers are separated from each other by at least 30 bases. Itis still more preferred that, when hybridized, the primers in a set ofprimers are separated from each other by at least 40 bases. It is stillmore preferred that, when hybridized, the primers in a set of primersare separated from each other by at least 50 bases.

It is preferred that, when hybridized, the primers in a set of primersare separated from each other by no more than about 500 bases. It ismore preferred that, when hybridized, the primers in a set of primersare separated from each other by no more than about 400 bases. It isstill more preferred that, when hybridized, the primers in a set ofprimers are separated from each other by no more than about 300 bases.It is still more preferred that, when hybridized, the primers in a setof primers are separated from each other by no more than about 200bases. Any combination of the preferred upper and lower limits ofseparation described above are specifically contemplated, including allintermediate ranges. The primers in a set of primers need not, whenhybridized, be separated from each other by the same number of bases. Itis preferred that, when hybridized, the primers in a set of primers areseparated from each other by about the same number of bases.

The optimal separation distance between primers will not be the same forall DNA polymerases, because this parameter is dependent on the netpolymerization rate. A processive DNA polymerase will have acharacteristic polymerization rate which may range from 5 to 300nucleotides per second, and may be influenced by the presence or absenceof accessory ssDNA binding proteins and helicases. In the case of anon-processive polymerase, the net polymerization rate will depend onthe enzyme concentration, because at higher concentrations there aremore re-initiation events and thus the net polymerization rate will beincreased. An example of a processive polymerase is φ29 DNA polymerase,which proceeds at 50 nucleotides per second. An example of anon-processive polymerase is Vent exo(−) DNA polymerase, which will giveeffective polymerization rates of 4 nucleotides per second at lowconcentration, or 16 nucleotides per second at higher concentrations.

To obtain an optimal yield in an MSDA reaction, the primer spacing ispreferably adjusted to suit the polymerase being used. Long primerspacing is preferred when using a polymerase with a rapid polymerizationrate. Shorter primer spacing is preferred when using a polymerase with aslower polymerization rate. The following assay can be used to determineoptimal spacing with any polymerase. The assay uses sets of primers,with each set made up of 5 left primers and 5 right primers. The sets ofprimers are designed to hybridize adjacent to the same target sequencewith each of the different sets of primers having a different primerspacing. The spacing is varied systematically between the sets ofprimers in increments of 25 nucleotides within the range of 25nucleotides to 400 nucleotides (the spacing of the primers within eachset is the same). A series of reactions are performed in which the sametarget sequence is amplified using the different sets of primers. Thespacing that produces the highest experimental yield of DNA is theoptimal primer spacing for the specific DNA polymerase, or DNApolymerase plus accessory protein combination being used.

DNA replication initiated at the sites in the target sequence where theprimers hybridize will extend to and displace strands being replicatedfrom primers hybridized at adjacent sites. Displacement of an adjacentstrand makes it available for hybridization to another primer andsubsequent initiation of another round of replication. The region(s) ofthe target sequence to which the primers hybridize is referred to as thehybridization target of the target sequence.

A set of primers can include any desired number of primers of differentnucleotide sequence. For MSDA, it is preferred that a set of primersinclude a plurality of primers. It is more preferred that a set ofprimers include 3 or more primers. It is still more preferred that a setof primers include 4 or more, 5 or more, 6 or more, or 7 or moreprimers. In general, the more primers used, the greater the level ofamplification that will be obtained. There is no fundamental upper limitto the number of primers that a set of primers can have. However, for agiven target sequence, the number of primers in a set of primers willgenerally be limited to the number of hybridization sites available inthe target sequence. For example, if the target sequence is a 10,000nucleotide DNA molecule and 20 nucleotide primers are used, there are500 non-overlapping 20 nucleotide sites in the target sequence. Evenmore primers than this could be used if overlapping sites are eitherdesired or acceptable. It is preferred that a set of primers include nomore than about 300 primers. It is preferred that a set of primersinclude no more than about 200 primers. It is still more preferred thata set of primers include no more than about 100 primers. It is morepreferred that a set of primers include no more than about 50 primers.It is most preferred that a set of primers include from 7 to about 50primers. Any combination of the preferred upper and lower limits for thenumber of primers in a set of primers described above are specificallycontemplated, including all intermediate ranges.

A preferred form of primer set for use in MSDA includes two sets ofprimers, referred to as a right set of primers and a left set ofprimers. The right set of primers and left set of primers are designedto be complementary to opposite strands of a target sequence. It ispreferred that the complementary portions of the right set of primersare each complementary to the right hybridization target, and that eachis complementary to a different portion of the right hybridizationtarget. It is preferred that the complementary portions of the left setof primers are each complementary to the left hybridization target, andthat each is complementary to a different portion of the lefthybridization target. The right and left hybridization targets flankopposite ends of the amplification target in a target sequence. It ispreferred that a right set of primers and a left set of primers eachinclude a preferred number of primers as described above for a set ofprimers. Specifically, it is preferred that a right or left set ofprimers include a plurality of primers. It is more preferred that aright or left set of primers include 3 or more primers. It is still morepreferred that a right or left set of primers include 4 or more, 5 ormore, 6 or more, or 7 or more primers. It is preferred that a right orleft set of primers include no more than about 200 primers. It is morepreferred that a right or left set of primers include no more than about100 primers. It is most preferred that a right or left set of primersinclude from 7 to about 100 primers. Any combination of the preferredupper and lower limits for the number of primers in a set of primersdescribed above are specifically contemplated, including allintermediate ranges. It is also preferred that, for a given targetsequence, the right set of primers and the left set of primers includethe same number of primers. It is also preferred that, for a giventarget sequence, the right set of primers and the left set of primersare composed of primers of similar length and/or hybridizationcharacteristics.

Where the target sequence(s) are present in mixed sample—for example, anosocomial sample containing both human and non-human nucleic acids—theprimers used can be specific for the nucleic acids of interest. Thus, todetect pathogen (that is, non-human) nucleic acids in a patient sample,primers specific to pathogen nucleic acids can be used. If human nucleicacids are to be detected, then primers specific to human nucleic acidscan be used. In this context, primers specific for particular targetnucleic acids or target sequences or a particular class of targetnucleic acids or target sequences refer to primers that supportamplification of the target nucleic acids and target sequences but donot support substantial amplification of non-target nucleic acids orsequences that are in the relevant sample.

3. Detection Tags

The non-complementary portion of a primer can include sequences to beused to further manipulate or analyze amplified sequences. An example ofsuch a sequence is a detection tag, which is a specific nucleotidesequence present in the non-complementary portion of a primer. Detectiontags have sequences complementary to detection probes. Detection tagscan be detected using their cognate detection probes. Detection tagsbecome incorporated at the ends of amplified strands. The result isamplified DNA having detection tag sequences that are complementary tothe complementary portion of detection probes. If present, there may beone, two, three, or more than three detection tags on a primer. It ispreferred that a primer have one, two, three or four detection tags.Most preferably, a primer will have one detection tag. Generally, it ispreferred that a primer have 10 detection tags or less. There is nofundamental limit to the number of detection tags that can be present ona primer except the size of the primer. When there are multipledetection tags, they may have the same sequence or they may havedifferent sequences, with each different sequence complementary to adifferent detection probe. It is preferred that a primer containdetection tags that have the same sequence such that they are allcomplementary to a single detection probe. For some multiplex detectionmethods, it is preferable that primers contain up to six detection tagsand that the detection tag portions have different sequences such thateach of the detection tag portions is complementary to a differentdetection probe. A similar effect can be achieved by using a set ofprimers where each has a single different detection tag. The detectiontags can each be any length that supports specific and stablehybridization between the detection tags and the detection probe. Forthis purpose, a length of 10 to 35 nucleotides is preferred, with adetection tag portion 15 to 20 nucleotides long being most preferred.

4. Address Tag

Another example of a sequence that can be included in thenon-complementary portion of a primer is an address tag. An address taghas a sequence complementary to an address probe. Address tags becomeincorporated at the ends of amplified strands. The result is amplifiedDNA having address tag sequences that are complementary to thecomplementary portion of address probes. If present, there may be one,or more than one, address tag on a primer. It is preferred that a primerhave one or two address tags. Most preferably, a primer will have oneaddress tag. Generally, it is preferred that a primer have 10 addresstags or less. There is no fundamental limit to the number of addresstags that can be present on a primer except the size of the primer. Whenthere are multiple address tags, they may have the same sequence or theymay have different sequences, with each different sequence complementaryto a different address probe. It is preferred that a primer containaddress tags that have the same sequence such that they are allcomplementary to a single address probe. The address tag portion can beany length that supports specific and stable hybridization between theaddress tag and the address probe. For this purpose, a length between 10and 35 nucleotides long is preferred, with an address tag portion 15 to20 nucleotides long being most preferred.

C. Lysis Solution

In the disclosed method, the cells can be exposed to alkaline conditionsby mixing the cells with a lysis solution. A lysis solution is generallya solution that can raise the pH of a cell solution sufficiently tocause cell lysis. Denaturing solutions can be used as lysis solutions solong as the denaturing solution can have the effects required of lysissolutions. In some embodiments, the lysis solution can comprises a base,such as an aqueous base. Useful bases include potassium hydroxide,sodium hydroxide, potassium acetate, sodium acetate, ammonium hydroxide,lithium hydroxide, calcium hydroxide, magnesium hydroxide, sodiumcarbonate, sodium bicarbonate, calcium carbonate, ammonia, aniline,benzylamine, n-butylamine, diethylamine, dimethylamine, diphenylamine,ethylamine, ethylenediamine, methylamine, N-methylaniline, morpholine,pyridine, triethylamine, trimethylamine, aluminum hydroxide, rubidiumhydroxide, cesium hydroxide, strontium hydroxide, barium hydroxide, andDBU (1,8-diazobicyclo[5,4,0]undec-7-ene). Useful formulations of lysissolution include lysis solution comprising 400 mM KOH, lysis solutioncomprising 400 mM KOH, 100 mM dithiothreitol, and 10 mM EDTA, and lysissolution consisting of 400 mM KOH, 100 mM dithiothreitol, and 10 mMEDTA.

In some embodiments, the lysis solution can comprise a plurality ofbasic agents. As used herein, a basic agent is a compound, compositionor solution that results in alkaline conditions. In some embodiments,the lysis solution can comprise a buffer. Useful buffers includephosphate buffers, “Good” buffers (such as BES, BICINE, CAPS, EPPS,HEPES, MES, MOPS, PIPES, TAPS, TES, and TRICINE), sodium cacodylate,sodium citrate, triethylammonium acetate, triethylammonium bicarbonate,Tris, Bis-tris, and Bis-tris propane. The lysis solution can comprise aplurality of buffering agents. As used herein, a buffering agent is acompound, composition or solution that acts as a buffer. An alkalinebuffering agent is a buffering agent that results in alkalineconditions. In some embodiments, the lysis solution can comprise acombination of one or more bases, basic agents, buffers and bufferingagents.

The amount of lysis solution mixed with the cells can be that amountthat causes a substantial number of cells to lyse or those that cause asufficient number of cells to lyse. Generally, this volume will be afunction of the pH of the cell/lysis solution mixture. Thus, the amountof lysis solution to mix with cells can be determined generally from thevolume of the cells and the alkaline concentration of the lysis buffer.For example, a smaller volume of a lysis solution with a stronger baseand/or higher concentration of base would be needed to create sufficientalkaline conditions than the volume needed of a lysis solution with aweaker base and/or lower concentration of base. The lysis solution canbe formulated such that the cells are mixed with an equal volume of thelysis solution (to produce the desired alkaline conditions).

For example, lysis solutions can be solutions that have a pH of fromabout 9.0 to about 13.0, from about 9.5 to about 13.0, from about 10.0to about 13.0, from about 10.5 to about 13.0, from about 11.0 to about13.0, from about 11.5 to about 13.0, from about 12.0 to about 13.0, fromabout 9.0 to about 12.0, from about 9.5 to about 12.0, from about 10.0to about 12.0, from about 10.5 to about 12.0, from about 11.0 to about12.0, from about 11.5 to about 12.0, from about 9.0 to about 11.5, fromabout 9.5 to about 11.5, from about 10.0 to about 11.5, from about 10.5to about 11.5, from about 11.0 to about 11.5, from about 9.0 to about11.0, from about 9.5 to about 11.0, from about 10.0 to about 11.0, fromabout 10.5 to about 11.0, from about 9.0 to about 10.5, from about 9.5to about 10.5, from about 10.0 to about 10.5, from about 9.0 to about10.0, from about 9.5 to about 10.0, from about 9.0 to about 9.5, about9.0, about 9.5, about 10.0, about 10.5, about 11.0, about 11.5, about12.0, about 12.5, or about 13.0.

Lysis solutions can have, for example, component concentrations of fromabout 10 mM to about 1 M, from about 10 mM to about 900 mM, from about10 mM to about 800 mM, from about 10 mM to about 700 mM, from about 10mM to about 600 mM, from about 10 mM to about 500 mM, from about 10 mMto about 400 mM, from about 10 mM to about 300 mM, from about 10 mM toabout 200 mM, from about 10 mM to about 100 mM, from about 10 mM toabout 90 mM, from about 10 mM to about 80 mM, from about 10 mM to about70 mM, from about 10 mM to about 60 mM, from about 10 mM to about 50 mM,from about 10 mM to about 40 mM, from about 10 mM to about 30 mM, fromabout 10 mM to about 20 mM, from about 20 mM to about 1 M, from about 20mM to about 900 mM, from about 20 mM to about 800 mM, from about 20 mMto about 700 mM, from about 20 mM to about 600 mM, from about 20 mM toabout 500 mM, from about 20 mM to about 400 mM, from about 20 mM toabout 300 mM, from about 20 mM to about 200 mM, from about 20 mM toabout 100 mM, from about 20 mM to about 90 mM, from about 20 mM to about80 mM, from about 20 mM to about 70 mM, from about 20 mM to about 60 mM,from about 20 mM to about 50 mM, from about 20 mM to about 40 mM, fromabout 20 mM to about 30 mM, from about 30 mM to about 1 M, from about 30mM to about 900 mM, from about 30 mM to about 800 mM, from about 30 mMto about 700 mM, from about 30 mM to about 600 mM, from about 30 mM toabout 500 mM, from about 30 mM to about 400 mM, from about 30 mM toabout 300 mM, from about 30 mM to about 200 mM, from about 30 mM toabout 100 mM, from about 30 mM to about 90 mM, from about 30 mM to about80 mM, from about 30 mM to about 70 mM, from about 30 mM to about 60 mM,from about 30 mM to about 50 mM, from about 30 mM to about 40 mM, fromabout 40 mM to about 1 M, from about 40 mM to about 900 mM, from about40 mM to about 800 mM, from about 40 mM to about 700 mM, from about 40mM to about 600 mM, from about 40 mM to about 500 mM, from about 40 mMto about 400 mM, from about 40 mM to about 300 mM, from about 40 mM toabout 200 mM, from about 40 mM to about 100 mM, from about 40 mM toabout 90 mM, from about 40 mM to about 80 mM, from about 40 mM to about70 mM, from about 40 mM to about 60 mM, from about 40 mM to about 50 mM,from about 50 mM to about 1 M, from about 50 mM to about 900 mM, fromabout 50 mM to about 800 mM, from about 50 mM to about 700 mM, fromabout 50 mM to about 600 mM, from about 50 mM to about 500 mM, fromabout 50 mM to about 400 mM, from about 50 mM to about 300 mM, fromabout 50 mM to about 200 mM, from about 50 mM to about 100 mM, fromabout 50 mM to about 90 mM, from about 50 mM to about 80 mM, from about50 mM to about 70 mM, from about 50 mM to about 60 mM, from about 60 mMto about 1 M, from about 60 mM to about 900 mM, from about 60 mM toabout 800 mM, from about 60 mM to about 700 mM, from about 60 mM toabout 600 mM, from about 60 mM to about 500 mM, from about 60 mM toabout 400 mM, from about 60 mM to about 300 mM, from about 60 mM toabout 200 mM, from about 60 mM to about 100 mM, from about 60 mM toabout 90 mM, from about 60 mM to about 80 mM, from about 60 mM to about70 mM, from about 70 mM to about 1 M, from about 70 mM to about 900 mM,from about 70 mM to about 800 mM, from about 70 mM to about 700 mM, fromabout 70 mM to about 600 mM, from about 70 mM to about 500 mM, fromabout 70 mM to about 400 mM, from about 70 mM to about 300 mM, fromabout 70 mM to about 200 mM, from about 70 mM to about 00 mM, from about70 mM to about 90 mM, from about 70 mM to about 80 mM, from about 80 mMto about 1 M, from about 80 mM to about 900 mM, from about 80 mM toabout 800 mM, from about 80 mM to about 700 mM, from about 80 mM toabout 600 mM, from about 80 mM to about 500 mM, from about 80 mM toabout 400 mM, from about 80 mM to about 300 mM, from about 80 mM toabout 200 mM, from about 80 mM to about 100 mM, from about 80 mM toabout 90 mM, from about 90 mM to about 1 M, from about 90 mM to about900 mM, from about 90 mM to about 800 mM, from about 90 mM to about 700mM, from about 90 mM to about 600 mM, from about 90 mM to about 500 mM,from about 90 mM to about 400 mM, from about 90 mM to about 300 mM, fromabout 90 mM to about 200 mM, from about 90 mM to about 100 mM, fromabout 100 mM to about 1 M, from about 100 mM to about 900 mM, from about100 mM to about 800 mM, from about 100 mM to about 700 mM, from about100 mM to about 600 mM, from about 100 mM to about 500 mM, from about100 mM to about 400 mM, from about 100 mM to about 300 mM, from about100 mM to about 200 mM, from about 200 mM to about 1 M, from about 200mM to about 900 mM, from about 200 mM to about 800 mM, from about 200 mMto about 700 mM, from about 200 mM to about 600 mM, from about 200 mM toabout 500 mM, from about 200 mM to about 400 mM, from about 200 mM toabout 300 mM, from about 300 mM to about 1 M, from about 300 mM to about900 mM, from about 300 mM to about 800 mM, from about 300 mM to about700 mM, from about 300 mM to about 600 mM, from about 300 mM to about500 mM, from about 300 mM to about 400 mM, from about 400 mM to about 1M, from about 400 mM to about 900 mM, from about 400 mM to about 800 mM,from about 400 mM to about 700 mM, from about 400 mM to about 600 mM,from about 400 mM to about 500 mM, from about 500 mM to about 1 M, fromabout 500 mM to about 900 mM, from about 500 mM to about 800 mM, fromabout 500 mM to about 700 mM, from about 500 mM to about 600 mM, fromabout 600 mM to about 1 M, from about 600 mM to about 900 mM, from about600 mM to about 800 mM, from about 600 mM to about 700 mM, from about700 mM to about 1 M, from about 700 mM to about 900 mM, from about 700mM to about 800 mM, from about 800 mM to about 1 M, from about 800 mM toabout 900 mM, from about 900 mM to about 1 M, about 10 mM, about 20 mM,about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about80 mM, about 90 mM, about 100 mM, about 200 mM, about 300 mM, about 400mM, about 500 mM, about 600 mM, about 700 mM, about 800 mM, about 900mM, or about 1 M.

The lysis solution can be composed of multiple solutions and/orcomponents that can be added to cells separately or combined indifferent combinations prior to addition to cells. Thus, for example, asolution of 400 mM KOH and 10 mM EDTA and a solution of 100 mMdithiothreitol can be added to the cells separately. Similarly, thedisclosed kits can be composed of multiple solutions and/or componentsto be combined to form a lysis solution prior to addition to cells orfor separate addition to cells.

D. Stabilization Solution

In the disclosed method, the pH of the cell lysate can be reduced toform a stabilized cell lysate. A stabilization solution is generally asolution that can reduce the pH of a cell lysate exposed to alkalineconditions as described elsewhere herein. In some embodiments, thestabilization solution can comprise an acid. Useful acids includehydrochloric acid, sulfuric acid, phosphoric acid, acetic acid,acetylsalicylic acid, ascorbic acid, carbonic acid, citric acid, formicacid, nitric acid, perchloric acid, HF, HBr, HI, H₂S, HCN, HSCN, HClO,monochloroacetic acid, dichloroacetic acid, trichloroacetic acid, andany carboxylic acid (ethanoic, propanoic, butanoic, etc., including bothlinear or branched chain carboxylic acids). In some embodiments, thestabilization solution can comprise a buffer. Useful buffers includeTris-HCl, HEPES, “Good” buffers (such as BES, BICINE, CAPS, EPPS, HEPES,MES, MOPS, PIPES, TAPS, TES, and TRICINE), sodium cacodylate, sodiumcitrate, triethylammonium acetate, triethylammonium bicarbonate, Tris,Bis-tris, and Bis-tris propane. Useful formulations of stabilizationsolutions include stabilization solution comprising 800 mM Tris-HCl;stabilization solution comprising 800 mM Tris-HCl at pH 4.1, andstabilization solution consisting of 800 mM Tris-HCl, pH 4.1.

In some embodiments, the stabilization solution can comprise a pluralityof acidic agents. As used herein, an acidic agent is a compound,composition or solution that forms an acid in solution. In someembodiments, the stabilization solution can comprise a plurality ofbuffering agents. An acidic buffering agent is a buffering agent thatforms an acid in solution. In some embodiments, the stabilizationsolution can comprise a combination of one or more acids, acidic agents,buffers and buffering agents.

A stabilized cell lysate is a cell lysate the pH of which is in theneutral range (from about pH 6.0 to about pH 9.0). Useful stabilizedcell lysates have a pH that allows replication of nucleic acids in thecell lysate. For example, the pH of the stabilized cell lysate isusefully at a pH at which the DNA polymerase can function. The pH of thecell lysate can be reduced by mixing the cell lysate with astabilization solution.

The amount of stabilization solution mixed with the cell lysate can bethat amount that causes a reduction in pH to the neutral range (or otherdesired pH value). Generally, this volume will be a function of the pHof the cell lysate/stabilization solution mixture. Thus, the amount ofstabilization solution to mix with the cell lysate can be determinedgenerally from the volume of the cell lysate, its pH and bufferingcapacity, and the acidic concentration of the stabilization buffer. Forexample, a smaller volume of a stabilization solution with a strongeracid and/or higher concentration of acid would be needed to reduce thepH sufficiently than the volume needed of a stabilization solution witha weaker acid and/or lower concentration of acid. The stabilizationsolution can be formulated such that the cell lysate is mixed with anequal volume of the stabilization solution (to produce the desired pH).

For example, stabilization solutions can be solutions that have a pH offrom about 1.0 to about 6.0, from about 2.0 to about 6.0, from about 3.0to about 6.0, from about 3.5 to about 6.0, from about 4.0 to about 6.0,from about 4.5 to about 6.0, from about 5.0 to about 6.0, from about 5.5to about 6.0, from about 1.0 to about 5.5, from about 2.0 to about 5.5,from about 3.0 to about 5.5, from about 3.5 to about 5.5, from about 4.0to about 5.5, from about 4.5 to about 5.5, from about 5.0 to about 5.5,from about 1.0 to about 5.0, from about 2.0 to about 5.0, from about 3.0to about 5.0, from about 3.5 to about 5.0, from about 4.0 to about 5.0,from about 4.5 to about 5.0, from about 1.0 to about 4.5, from about 2.0to about 4.5, from about 3.0 to about 4.5, from about 3.5 to about 4.5,from about 4.0 to about 4.5, from about 1.0 to about 4.0, from about 2.0to about 4.0, from about 3.0 to about 4.0, from about 3.5 to about 4.0,from about 1.0 to about 3.5, from about 2.0 to about 3.5, from about 3.0to about 3.5, from about 1.0 to about 3.0, from about 2.0 to about 3.0,from about 1.0 to about 2.5, from about 2.0 to about 2.5, from about 1.0to about 2.0, about 1.0, about 2.0, about 2.5, about 3.0, about 3.5,about 4.0, about 4.5, about 5.0, about 5.5, or about 6.0.

Stabilization solutions can have, for example, component concentrationsof from about 100 mM to about 1 M, from about 100 mM to about 900 mM,from about 100 mM to about 800 mM, from about 100 mM to about 700 mM,from about 100 mM to about 600 mM, from about 100 mM to about 500 mM,from about 100 mM to about 400 mM, from about 100 mM to about 300 mM,from about 100 mM to about 200 mM, from about 200 mM to about 1 M, fromabout 200 mM to about 900 mM, from about 200 mM to about 800 mM, fromabout 200 mM to about 700 mM, from about 200 mM to about 600 mM, fromabout 200 mM to about 500 mM, from about 200 mM to about 400 mM, fromabout 200 mM to about 300 mM, from about 300 mM to about 1 M, from about300 mM to about 900 mM, from about 300 mM to about 800 mM, from about300 mM to about 700 mM, from about 300 mM to about 600 mM, from about300 mM to about 500 mM, from about 300 mM to about 400 mM, from about400 mM to about 1 M, from about 400 mM to about 900 mM, from about 400mM to about 800 mM, from about 400 mM to about 700 mM, from about 400 mMto about 600 mM, from about 400 mM to about 500 mM, from about 500 mM toabout 1 M, from about 500 mM to about 900 mM, from about 500 mM to about800 mM, from about 500 mM to about 700 mM, from about 500 mM to about600 mM, from about 600 mM to about 1 M, from about 600 mM to about 900mM, from about 600 mM to about 800 mM, from about 600 mM to about 700mM, from about 700 mM to about 1 M, from about 700 mM to about 900 mM,from about 700 mM to about 800 mM, from about 800 mM to about 1 M, fromabout 800 mM to about 900 mM, from about 900 mM to about 1 M, about 100mM, about 200 mM, about 300 mM, about 400 mM, about 500 mM, about 600mM, about 700 mM, about 800 mM, about 900 mM, or about 1 M.

The stabilization solution can be composed of multiple solutions and/orcomponents that can be added to cell lysates separately or combined indifferent combinations prior to addition to cell lysates. Thus, forexample, a solution of a buffer and a solution of an acid can be addedto the cells separately. Similarly, the disclosed kits can be composedof multiple solutions and/or components to be combined to form astabilization solution prior to addition to cell lysates or for separateaddition to cell lysates.

E. Denaturing Solution

In some forms of the disclosed method, the DNA samples can be exposed todenaturing conditions by mixing the sample with a denaturing solution. Adenaturing solution is generally a solution that can raise the pH of asample sufficiently to cause, in combination with other conditions suchas heating, substantial denaturation of DNA in the DNA sample.Substantial denaturation refers to denaturation of 90% or more of thenucleotides in 90% or more of the DNA molecules in a sample. In thiscontext, denaturation of nucleotides refers to unpaired nucleotideswhether physically denatured by treatment or already unpaired in thesample. Lysis solutions can be used as denaturing solutions so long asthe lysis solution has the effects required of denaturing solutions.

In some embodiments, the denaturing solution can comprises a base, suchas an aqueous base. Useful bases include potassium hydroxide, sodiumhydroxide, potassium acetate, sodium acetate, ammonium hydroxide,lithium hydroxide, calcium hydroxide, magnesium hydroxide, sodiumcarbonate, sodium bicarbonate, calcium carbonate, ammonia, aniline,benzylamine, n-butylamine, diethylamine, dimethylamine, diphenylamine,ethylamine, ethylenediamine, methylamine, N-methylaniline, morpholine,pyridine, triethylamine, trimethylamine, aluminum hydroxide, rubidiumhydroxide, cesium hydroxide, strontium hydroxide, barium hydroxide, andDBU (1,8-diazobicyclo[5,4,0]undec-7-ene). Useful formulations ofdenaturing solution include denaturing solution comprising about 150 mMto about 500 mM NaOH, denaturing solution comprising about 150 mM toabout 500 mM NaOH, and denaturing solution consisting of about 150 mM toabout 500 mM NaOH.

In some embodiments, the denaturing solution can comprise a plurality ofbasic agents. As used herein, a basic agent is a compound, compositionor solution that results in denaturing conditions. In some embodiments,the denaturing solution can comprise a buffer. Useful buffers includephosphate buffers, “Good” buffers (such as BES, BICINE, CAPS, EPPS,HEPES, MES, MOPS, PIPES, TAPS, TES, and TRICINE), sodium cacodylate,sodium citrate, triethylammonium acetate, triethylammonium bicarbonate,Tris, Bis-tris, and Bis-tris propane. The denaturing solution cancomprise a plurality of buffering agents. As used herein, a bufferingagent is a compound, composition or solution that acts as a buffer. Analkaline buffering agent is a buffering agent that results in alkalineconditions. In some embodiments, the denaturing solution can comprise acombination of one or more bases, basic agents, buffers and bufferingagents.

The amount of denaturing solution mixed with the DNA samples can be thatamount that causes, in combination with other conditions such asheating, substantial denaturation of DNA in the DNA sample. Generally,this volume will be a function of the pH, ionic strength, andtemperature of the sample/denaturing solution mixture. Thus, the amountof denaturing solution to mix with DNA samples can be determinedgenerally from the volume of the DNA sample, the alkaline concentrationof the denaturing buffer, and the temperature to which the resultingmixture will be heated. For example, at a given temperature, a smallervolume of a denaturing solution with a stronger base and/or higherconcentration of base would be needed to create sufficient denaturingconditions than the volume needed of a denaturing solution with a weakerbase and/or lower concentration of base. The denaturing solution can beformulated such that the DNA samples are mixed with, for example, onetenth volume of the denaturing solution (to produce the desireddenaturing conditions).

For example, denaturing solutions can be solutions that have a pH offrom about 9.0 to about 13.0, from about 9.5 to about 13.0, from about10.0 to about 13.0, from about 10.5 to about 13.0, from about 11.0 toabout 13.0, from about 11.5 to about 13.0, from about 12.0 to about13.0, from about 9.0 to about 12.0, from about 9.5 to about 12.0, fromabout 10.0 to about 12.0, from about 10.5 to about 12.0, from about 11.0to about 12.0, from about 11.5 to about 12.0, from about 9.0 to about11.5, from about 9.5 to about 11.5, from about 10.0 to about 11.5, fromabout 10.5 to about 11.5, from about 11.0 to about 11.5, from about 9.0to about 11.0, from about 9.5 to about 11.0, from about 10.0 to about11.0, from about 10.5 to about 11.0, from about 9.0 to about 10.5, fromabout 9.5 to about 10.5, from about 10.0 to about 10.5, from about 9.0to about 10.0, from about 9.5 to about 10.0, from about 9.0 to about9.5, about 9.0, about 9.5, about 10.0, about 10.5, about 1.0, about11.5, about 12.0, about 12.5, or about 13.0.

Denaturing solutions can have, for example, component concentrations offrom about 10 mM to about 1 M, from about 10 mM to about 900 mM, fromabout 10 mM to about 800 mM, from about 10 mM to about 700 mM, fromabout 10 mM to about 600 mM, from about 10 mM to about 500 mM, fromabout 10 mM to about 400 mM, from about 10 mM to about 300 mM, fromabout 10 mM to about 200 mM, from about 10 mM to about 100 mM, fromabout 10 mM to about 90 mM, from about 10 mM to about 80 mM, from about10 mM to about 70 mM, from about 10 mM to about 60 mM, from about 10 mMto about 50 mM, from about 10 mM to about 40 mM, from about 10 mM toabout 30 mM, from about 10 mM to about 20 mM, from about 20 mM to about1 M, from about 20 mM to about 900 mM, from about 20 mM to about 800 mM,from about 20 mM to about 700 mM, from about 20 mM to about 600 mM, fromabout 20 mM to about 500 mM, from about 20 mM to about 400 mM, fromabout 20 mM to about 300 mM, from about 20 mM to about 200 mM, fromabout 20 mM to about 100 mM, from about 20 mM to about 90 mM, from about20 mM to about 80 mM, from about 20 mM to about 70 mM, from about 20 mMto about 60 mM, from about 20 mM to about 50 mM, from about 20 mM toabout 40 mM, from about 20 mM to about 30 mM, from about 30 mM to about1 M, from about 30 mM to about 900 mM, from about 30 mM to about 800 mM,from about 30 mM to about 700 mM, from about 30 mM to about 600 mM, fromabout 30 mM to about 500 mM, from about 30 mM to about 400 mM, fromabout 30 mM to about 300 mM, from about 30 mM to about 200 mM, fromabout 30 mM to about 100 mM, from about 30 mM to about 90 mM, from about30 mM to about 80 mM, from about 30 mM to about 70 mM, from about 30 mMto about 60 mM, from about 30 mM to about 50 mM, from about 30 mM toabout 40 mM, from about 40 mM to about 1 M, from about 40 mM to about900 mM, from about 40 mM to about 800 mM, from about 40 mM to about 700mM, from about 40 mM to about 600 mM, from about 40 mM to about 500 mM,from about 40 mM to about 400 mM, from about 40 mM to about 300 mM, fromabout 40 mM to about 200 mM, from about 40 mM to about 100 mM, fromabout 40 mM to about 90 mM, from about 40 mM to about 80 mM, from about40 mM to about 70 mM, from about 40 mM to about 60 mM, from about 40 mMto about 50 mM, from about 50 mM to about 1 M, from about 50 mM to about900 mM, from about 50 mM to about 800 mM, from about 50 mM to about 700mM, from about 50 mM to about 600 mM, from about 50 mM to about 500 mM,from about 50 mM to about 400 mM, from about 50 mM to about 300 mM, fromabout 50 mM to about 200 mM, from about 50 mM to about 100 mM, fromabout 50 mM to about 90 mM, from about 50 mM to about 80 mM, from about50 mM to about 70 mM, from about 50 mM to about 60 mM, from about 60 mMto about 1 M, from about 60 mM to about 900 mM, from about 60 mM toabout 800 mM, from about 60 mM to about 700 mM, from about 60 mM toabout 600 mM, from about 60 mM to about 500 mM, from about 60 mM toabout 400 mM, from about 60 mM to about 300 mM, from about 60 mM toabout 200 mM, from about 60 mM to about 100 mM, from about 60 mM toabout 90 mM, from about 60 mM to about 80 mM, from about 60 mM to about70 mM, from about 70 mM to about 1 M, from about 70 mM to about 900 mM,from about 70 mM to about 800 mM, from about 70 mM to about 700 mM, fromabout 70 mM to about 600 mM, from about 70 mM to about 500 mM, fromabout 70 mM to about 400 mM, from about 70 mM to about 300 mM, fromabout 70 mM to about 200 mM, from about 70 mM to about 100 mM, fromabout 70 mM to about 90 mM, from about 70 mM to about 80 mM, from about80 mM to about 1 M, from about 80 mM to about 900 mM, from about 80 mMto about 800 mM, from about 80 mM to about 700 mM, from about 80 mM toabout 600 mM, from about 80 mM to about 500 mM, from about 80 mM toabout 400 mM, from about 80 mM to about 300 mM, from about 80 mM toabout 200 mM, from about 80 mM to about 100 mM, from about 80 mM toabout 90 mM, from about 90 mM to about 1 M, from about 90 mM to about900 mM, from about 90 mM to about 800 mM, from about 90 mM to about 700mM, from about 90 mM to about 600 mM, from about 90 mM to about 500 mM,from about 90 mM to about 400 mM, from about 90 mM to about 300 mM, fromabout 90 mM to about 200 mM, from about 90 mM to about 100 mM, fromabout 100 mM to about 1 M, from about 100 mM to about 900 mM, from about100 mM to about 800 mM, from about 100 mM to about 700 mM, from about 00mM to about 600 mM, from about 100 mM to about 500 mM, from about 100 mMto about 400 mM, from about 100 mM to about 300 mM, from about 00 mM toabout 200 mM, from about 200 mM to about 1 M, from about 200 mM to about900 mM, from about 200 mM to about 800 mM, from about 200 mM to about700 mM, from about 200 mM to about 600 mM, from about 200 mM to about500 mM, from about 200 mM to about 400 mM, from about 200 mM to about300 mM, from about 300 mM to about 1 M, from about 300 mM to about 900mM, from about 300 mM to about 800 mM, from about 300 mM to about 700mM, from about 300 mM to about 600 mM, from about 300 mM to about 500mM, from about 300 mM to about 400 mM, from about 400 mM to about 1 M,from about 400 mM to about 900 mM, from about 400 mM to about 800 mM,from about 400 mM to about 700 mM, from about 400 mM to about 600 mM,from about 400 mM to about 500 mM, from about 500 mM to about 1 M, fromabout 500 mM to about 900 mM, from about 500 mM to about 800 mM, fromabout 500 mM to about 700 mM, from about 500 mM to about 600 mM, fromabout 600 mM to about 1 M, from about 600 mM to about 900 mM, from about600 mM to about 800 mM, from about 600 mM to about 700 mM, from about700 mM to about 1 M, from about 700 mM to about 900 mM, from about 700mM to about 800 mM, from about 800 mM to about 1 M, from about 800 mM toabout 900 mM, from about 900 mM to about 1 M, about 10 mM, about 20 mM,about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about80 mM, about 90 mM, about 100 mM, about 200 mM, about 300 mM, about 400mM, about 500 mM, about 600 mM, about 700 mM, about 800 mM, about 900mM, or about 1 M.

The denaturing solution can be composed of multiple solutions and/orcomponents that can be added to DNA samples separately or combined indifferent combinations prior to addition to DNA samples. Thus, forexample, a solution of a buffer and a solution of a base can be added tothe samples separately. Similarly, the disclosed kits can be composed ofmultiple solutions and/or components to be combined to form a denaturingsolution prior to addition to DNA samples or for separate addition tosamples.

F. Nucleic Acid Fingerprints

The disclosed method can be used to produce replicated strands thatserve as a nucleic acid fingerprint of a complex sample of nucleic acid.Such a nucleic acid fingerprint can be compared with other, similarlyprepared nucleic acid fingerprints of other nucleic acid samples toallow convenient detection of differences between the samples. Thenucleic acid fingerprints can be used both for detection of relatednucleic acid samples and comparison of nucleic acid samples. Forexample, the presence or identity of specific organisms can be detectedby producing a nucleic acid fingerprint of the test organism andcomparing the resulting nucleic acid fingerprint with reference nucleicacid fingerprints prepared from known organisms. Changes and differencesin gene expression patterns can also be detected by preparing nucleicacid fingerprints of mRNA from different cell samples and comparing thenucleic acid fingerprints. The replicated strands can also be used toproduce a set of probes or primers that is specific for the source of anucleic acid sample. The replicated strands can also be used as alibrary of nucleic acid sequences present in a sample. Nucleic acidfingerprints can be made up of, or derived from, whole genomeamplification of a sample such that the entire relevant nucleic acidcontent of the sample is substantially represented, or from multiplestrand displacement amplification of selected target sequences within asample.

Nucleic acid fingerprints can be stored or archived for later use. Forexample, replicated strands produced in the disclosed method can bephysically stored, either in solution, frozen, or attached or adhered toa solid-state substrate such as an array. Storage in an array is usefulfor providing an archived probe set derived from the nucleic acids inany sample of interest. As another example, informational content of, orderived from, nucleic acid fingerprints can also be stored. Suchinformation can be stored, for example, in or as computer readablemedia. Examples of informational content of nucleic acid fingerprintsinclude nucleic acid sequence information (complete or partial);differential nucleic acid sequence information such as sequences presentin one sample but not another; hybridization patterns of replicatedstrands to, for example, nucleic acid arrays, sets, chips, or otherreplicated strands. Numerous other data that is or can be derived fromnucleic acid fingerprints and replicated strands produced in thedisclosed method can also be collected, used, saved, stored, and/orarchived.

Nucleic acid fingerprints can also contain or be made up of otherinformation derived from the information generated in the disclosedmethod, and can be combined with information obtained or generated fromany other source. The informational nature of nucleic acid fingerprintsproduced using the disclosed method lends itself to combination and/oranalysis using known bioinformatics systems and methods.

Nucleic acid fingerprints of nucleic acid samples can be compared to asimilar nucleic acid fingerprint derived from any other sample to detectsimilarities and differences in the samples (which is indicative ofsimilarities and differences in the nucleic acids in the samples). Forexample, a nucleic acid fingerprint of a first nucleic acid sample canbe compared to a nucleic acid fingerprint of a sample from the same typeof organism as the first nucleic acid sample, a sample from the sametype of tissue as the first nucleic acid sample, a sample from the sameorganism as the first nucleic acid sample, a sample obtained from thesame source but at time different from that of the first nucleic acidsample, a sample from an organism different from that of the firstnucleic acid sample, a sample from a type of tissue different from thatof the first nucleic acid sample, a sample from a strain of organismdifferent from that of the first nucleic acid sample, a sample from aspecies of organism different from that of the first nucleic acidsample, or a sample from a type of organism different from that of thefirst nucleic acid sample.

The same type of tissue is tissue of the same type such as liver tissue,muscle tissue, or skin (which may be from the same or a differentorganism or type of organism). The same organism refers to the sameindividual, animal, or cell. For example, two samples taken from apatient are from the same organism. The same source is similar butbroader, referring to samples from, for example, the same organism, thesame tissue from the same organism, the same DNA molecule, or the sameDNA library. Samples from the same source that are to be compared can becollected at different times (thus allowing for potential changes overtime to be detected). This is especially useful when the effect of atreatment or change in condition is to be assessed. Samples from thesame source that have undergone different treatments can also becollected and compared using the disclosed method. A different organismrefers to a different individual organism, such as a different patient,a different individual animal. Different organism includes a differentorganism of the same type or organisms of different types. A differenttype of organism refers to organisms of different types such as a dogand cat, a human and a mouse, or E. coli and Salmonella. A differenttype of tissue refers to tissues of different types such as liver andkidney, or skin and brain. A different strain or species of organismrefers to organisms differing in their species or strain designation asthose terms are understood in the art.

G. Solid-State Detectors

Solid-state detectors are solid-state substrates or supports to whichaddress probes or detection molecules have been coupled. A preferredform of solid-state detector is an array detector. An array detector isa solid-state detector to which multiple different address probes ordetection molecules have been coupled in an array, grid, or otherorganized pattern.

Solid-state substrates for use in solid-state detectors can include anysolid material to which oligonucleotides can be coupled. This includesmaterials such as acrylamide, cellulose, nitrocellulose, glass, gold,polystyrene, polyethylene vinyl acetate, polypropylene,polymethacrylate, polyethylene, polyethylene oxide, glass,polysilicates, polycarbonates, teflon, fluorocarbons, nylon, siliconrubber, polyanhydrides, polyglycolic acid, polylactic acid,polyorthoesters, functionalized silane, polypropylfumerate, collagen,glycosaminoglycans, and polyamino acids. Solid-state substrates can haveany useful form including thin films or membranes, beads, bottles,dishes, fibers, optical fibers, woven fibers, chips, compact disks,shaped polymers, particles and microparticles. A chip is a rectangularor square small piece of material. Preferred forms for solid-statesubstrates are thin films, beads, or chips.

Address probes immobilized on a solid-state substrate allow capture ofthe products of the disclosed amplification method on a solid-statedetector. Such capture provides a convenient means of washing awayreaction components that might interfere with subsequent detectionsteps. By attaching different address probes to different regions of asolid-state detector, different amplification products can be capturedat different, and therefore diagnostic, locations on the solid-statedetector. For example, in a multiplex assay, address probes specific fornumerous different amplified nucleic acids (each representing adifferent target sequence amplified via a different set of primers) canbe immobilized in an array, each in a different location. Capture anddetection will occur only at those array locations corresponding toamplified nucleic acids for which the corresponding target sequenceswere present in a sample.

Methods for immobilization of oligonucleotides to solid-state substratesare well established. Oligonucleotides, including address probes anddetection probes, can be coupled to substrates using establishedcoupling methods. For example, suitable attachment methods are describedby Pease et al., Proc. Natl. Acad. Sci. USA 91(11):5022–5026 (1994), andKhrapko et al., Mol Biol (Mosk) (USSR) 25:718–730 (1991). A method forimmobilization of 3′-amine oligonucleotides on casein-coated slides isdescribed by Stimpson et al., Proc. Natl. Acad. Sci. USA 92:6379–6383(1995). A preferred method of attaching oligonucleotides to solid-statesubstrates is described by Guo et al., Nucleic Acids Res. 22:5456–5465(1994). Examples of nucleic acid chips and arrays, including methods ofmaking and using such chips and arrays, are described in U.S. Pat. Nos.6,287,768, 6,288,220, 6,287,776, 6,297,006, and 6,291,193.

H. Solid-State Samples

Solid-state samples are solid-state substrates or supports to whichtarget sequences or MDA products (that is, replicated strands) have beencoupled or adhered. Target sequences are preferably delivered in atarget sample or assay sample. A preferred form of solid-state sample isan array sample. An array sample is a solid-state sample to whichmultiple different target sequences have been coupled or adhered in anarray, grid, or other organized pattern.

Solid-state substrates for use in solid-state samples can include anysolid material to which target sequences can be coupled or adhered. Thisincludes materials such as acrylamide, cellulose, nitrocellulose, glass,gold, polystyrene, polyethylene vinyl acetate, polypropylene,polymethacrylate, polyethylene, polyethylene oxide, glass,polysilicates, polycarbonates, teflon, fluorocarbons, nylon, siliconrubber, polyanhydrides, polyglycolic acid, polylactic acid,polyorthoesters, functionalized silane, polypropylfumerate, collagen,glycosaminoglycans, and polyamino acids. Solid-state substrates can haveany useful form including thin films or membranes, beads, bottles,dishes, slides, fibers, optical fibers, woven fibers, chips, compactdisks, shaped polymers, particles and microparticles. A chip is arectangular or square small piece of material. Preferred forms forsolid-state substrates are thin films, beads, or chips.

Target sequences immobilized on a solid-state substrate allow formationof target-specific amplified nucleic acid localized on the solid-statesubstrate. Such localization provides a convenient means of washing awayreaction components that might interfere with subsequent detectionsteps, and a convenient way of assaying multiple different samplessimultaneously. Amplified nucleic acid can be independently formed ateach site where a different sample is adhered. For immobilization oftarget sequences or other oligonucleotide molecules to form asolid-state sample, the methods described above can be used. Nucleicacids produced in the disclosed method can be coupled or adhered to asolid-state substrate in any suitable way. For example, nucleic acidsgenerated by multiple strand displacement can be attached by addingmodified nucleotides to the 3′ ends of nucleic acids produced by stranddisplacement replication using terminal deoxynucleotidyl transferase,and reacting the modified nucleotides with a solid-state substrate orsupport thereby attaching the nucleic acids to the solid-state substrateor support.

A preferred form of solid-state substrate is a glass slide to which upto 256 separate target samples have been adhered as an array of smalldots. Each dot is preferably from 0.1 to 2.5 mm in diameter, and mostpreferably around 2.5 mm in diameter. Such microarrays can befabricated, for example, using the method described by Schena et al.,Science 270:487–470 (1995). Briefly, microarrays can be fabricated onpoly-L-lysine-coated microscope slides (Sigma) with an arraying machinefitted with one printing tip. The tip is loaded with 1 μl of a DNAsample (0.5 mg/ml) from, for example, 96-well microtiter plates anddeposited ˜0.005 μl per slide on multiple slides at the desired spacing.The printed slides can then be rehydrated for 2 hours in a humidchamber, snap-dried at 100° C. for 1 minute, rinsed in 0.1% SDS, andtreated with 0.05% succinic anhydride prepared in buffer consisting of50% 1-methyl-2-pyrrolidinone and 50% boric acid. The DNA on the slidescan then be denatured in, for example, distilled water for 2 minutes at90° C. immediately before use. Microarray solid-state samples canscanned with, for example, a laser fluorescent scanner with acomputer-controlled XY stage and a microscope objective. A mixed gas,multiline laser allows sequential excitation of multiple fluorophores.

I. Detection Labels

To aid in detection and quantitation of nucleic acids amplified usingthe disclosed method, detection labels can be directly incorporated intoamplified nucleic acids or can be coupled to detection molecules. Asused herein, a detection label is any molecule that can be associatedwith amplified nucleic acid, directly or indirectly, and which resultsin a measurable, detectable signal, either directly or indirectly. Manysuch labels for incorporation into nucleic acids or coupling to nucleicacid probes are known to those of skill in the art. Examples ofdetection labels suitable for use in the disclosed method areradioactive isotopes, fluorescent molecules, phosphorescent molecules,enzymes, antibodies, and ligands.

Examples of suitable fluorescent labels include fluoresceinisothiocyanate (FITC), 5,6-carboxymethyl fluorescein, Texas red,nitrobenz-2-oxa-1,3-diazol-4-yl (NBD), coumarin, dansyl chloride,rhodamine, amino-methyl coumarin (AMCA), Eosin, Erythrosin, BODIPY®,Cascade Blue®, Oregon Green®, pyrene, lissamine, xanthenes; acridines,oxazines, phycoerythrin, macrocyclic chelates of lanthanide ions such asquantum dye™, fluorescent energy transfer dyes, such as thiazoleorange-ethidium heterodimer, and the cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5and Cy7. Examples of other specific fluorescent labels include3-Hydroxypyrene 5,8,10-Tri Sulfonic acid, 5-Hydroxy Tryptamine (5-HT),Acid Fuchsin, Alizarin Complexon, Alizarin Red, Allophycocyanin,Aminocoumarin, Anthroyl Stearate, Astrazon Brilliant Red 4G, AstrazonOrange R, Astrazon Red 6B, Astrazon Yellow 7 GLL, Atabrine, Auramine,Aurophosphine, Aurophosphine G, BAO 9 (Bisaminophenyloxadiazole), BCECF,Berberine Sulphate, Bisbenzamide, Blancophor FFG Solution, BlancophorSV, Bodipy F1, Brilliant Sulphoflavin FF, Calcien Blue, Calcium Green,Calcofluor RW Solution, Calcofluor White, Calcophor White ABT Solution,Calcophor White Standard Solution, Carbostyryl, Cascade Yellow,Catecholamine, Chinacrine, Coriphosphine O, Coumarin-Phalloidin, CY3.18, CY5.1 8, CY7, Dans (1-Dimethyl Amino Naphaline 5 Sulphonic Acid),Dansa (Diamino Naphtyl Sulphonic Acid), Dansyl NH-CH3, Diamino PhenylOxydiazole (DAO), Dimethylamino-5-Sulphonic acid, DipyrrometheneboronDifluoride, Diphenyl Brilliant Flavine 7GFF, Dopamine, Erythrosin ITC,Euchrysin, FIF (Formaldehyde Induced Fluorescence), Flazo Orange, Fluo3, Fluorescamine, Fura-2, Genacryl Brilliant Red B, Genacryl BrilliantYellow 10GF, Genacryl Pink 3G, Genacryl Yellow 5GF, Gloxalic Acid,Granular Blue, Haematoporphyrin, Indo-1, Intrawhite Cf Liquid, LeucophorPAF, Leucophor SF, Leucophor WS, Lissamine Rhodamine B200 (RD200),Lucifer Yellow CH, Lucifer Yellow VS, Magdala Red, Marina Blue, MaxilonBrilliant Flavin 10 GFF, Maxilon Brilliant Flavin 8 GFF, MPS (MethylGreen Pyronine Stilbene), Mithramycin, NBD Amine, Nitrobenzoxadidole,Noradrenaline, Nuclear Fast Red, Nuclear Yellow, Nylosan BrilliantFlavin E8G, Oxadiazole, Pacific Blue, Pararosaniline (Feulgen), PhorwiteAR Solution, Phorwite BKL, Phorwite Rev, Phorwite RPA, Phosphine 3R,Phthalocyanine, Phycoerythrin R, Polyazaindacene Pontochrome Blue Black,Porphyrin, Primuline, Procion Yellow, Pyronine, Pyronine B, PyrozalBrilliant Flavin 7GF, Quinacrine Mustard, Rhodamine 123, Rhodamine 5GLD, Rhodamine 6G, Rhodamine B, Rhodamine B 200, Rhodamine B Extra,Rhodamine BB, Rhodamine BG, Rhodamine WT, Serotonin, Sevron BrilliantRed 2B, Sevron Brilliant Red 4G, Sevron Brilliant Red B, Sevron Orange,Sevron Yellow L; SITS (Primuline), SITS (Stilbene Isothiosulphonicacid), Stilbene, Snarf 1, sulpho Rhodamine B Can C, Sulpho Rhodamine GExtra, Tetracycline, Thiazine Red R, Thioflavin S, Thioflavin TCN,Thioflavin 5, Thiolyte, Thiozol Orange, Tinopol CBS, True Blue,Ultralite, Uranine B, Uvitex SFC, Xylene Orange, and XRITC.

Preferred fluorescent labels are fluorescein(5-carboxyfluorescein-N-hydroxysuccinimide ester), rhodamine(5,6-tetramethyl rhodamine), and the cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5and Cy7. The absorption and emission maxima, respectively, for thesefluors are: FITC (490 nm; 520 nm), Cy3 (554 nm; 568 nm), Cy3.5 (581 nm;588 nm), Cy5 (652 nm: 672 nm), Cy5.5 (682 nm; 703 nm) and Cy7 (755 nm;778 nm), thus allowing their simultaneous detection. Other examples offluorescein dyes include 6-carboxyfluorescein (6-FAM),2′,4′,1,4,-tetrachlorofluorescein (TET),2′,4′,5′,7′,1,4-hexachlorofluorescein (HEX), 2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyrhodamine (JOE), 2′-chloro-5′-fluoro-7′,8′-fusedphenyl-1,4-dichloro-6-carboxyfluorescein (NED), and2′-chloro-7′-phenyl-1,4-dichloro-6-carboxyfluorescein (VIC). Fluorescentlabels can be obtained from a variety of commercial sources, includingAmersham Pharmacia Biotech, Piscataway, N.J.; Molecular Probes, Eugene,Oreg.; and Research Organics, Cleveland, Ohio.

Additional labels of interest include those that provide for signal onlywhen the probe with which they are associated is specifically bound to atarget molecule, where such labels include: “molecular beacons” asdescribed in Tyagi & Kramer, Nature Biotechnology (1996) 14:303 and EP 0070 685 B1. Other labels of interest include those described in U.S.Pat. No. 5,563,037; WO 97/17471 and WO 97/17076.

Labeled nucleotides are a preferred form of detection label since theycan be directly incorporated into the amplification products duringsynthesis. Examples of detection labels that can be incorporated intoamplified nucleic acids include nucleotide analogs such as BrdUrd(5-bromodeoxyuridine, Hoy and Schimke, Mutation Research 290:217–230(1993)), aminoallyldeoxyuridine (Henegariu et al., Nature Biotechnology18:345–348 (2000)), 5-methylcytosine (Sano et al., Biochim. Biophys.Acta 951:157–165 (1988)), bromouridine (Wansick et al., J. Cell Biology122:283–293 (1993)) and nucleotides modified with biotin (Langer et al.,Proc. Natl. Acad. Sci. USA 78:6633 (1981)) or with suitable haptens suchas digoxygenin (Kerkhof, Anal. Biochem. 205:359–364 (1992)). Suitablefluorescence-labeled nucleotides are Fluorescein-isothiocyanate-dUTP,Cyanine-3-dUTP and Cyanine-5-dUTP (Yu et al., Nucleic Acids Res.,22:3226–3232 (1994)). A preferred nucleotide analog detection label forDNA is BrdUrd (bromodeoxyuridine, BrdUrd, BrdU, BUdR, Sigma-Aldrich Co).Other preferred nucleotide analogs for incorporation of detection labelinto DNA are AA-dUTP (aminoallyl-deoxyuridine triphosphate,Sigma-Aldrich Co.), and 5-methyl-dCTP (Roche Molecular Biochemicals). Apreferred nucleotide analog for incorporation of detection label intoRNA is biotin-16-UTP (biotin-16-uridine-5′-triphosphate, Roche MolecularBiochemicals). Fluorescein, Cy3, and Cy5 can be linked to dUTP fordirect labelling. Cy3.5 and Cy7 are available as avidin oranti-digoxygenin conjugates for secondary detection of biotin- ordigoxygenin-labelled probes.

Detection labels that are incorporated into amplified nucleic acid, suchas biotin, can be subsequently detected using sensitive methodswell-known in the art. For example, biotin can be detected usingstreptavidin-alkaline phosphatase conjugate (Tropix, Inc.), which isbound to the biotin and subsequently detected by chemiluminescence ofsuitable substrates (for example, chemiluminescent substrate CSPD:disodium,3-(4-methoxyspiro-[1,2,-dioxetane-3–2′-(5′-chloro)tricyclo[3.3.1.1^(3,7)]decane]-4-yl)phenyl phosphate; Tropix, Inc.). Labels can also be enzymes, such asalkaline phosphatase, soybean peroxidase, horseradish peroxidase andpolymerases, that can be detected, for example, with chemical signalamplification or by using a substrate to the enzyme which produces light(for example, a chemiluminescent 1,2-dioxetane substrate) or fluorescentsignal.

Molecules that combine two or more of these detection labels are alsoconsidered detection labels. Any of the known detection labels can beused with the disclosed probes, tags, and method to label and detectnucleic acid amplified using the disclosed method. Methods for detectingand measuring signals generated by detection labels are also known tothose of skill in the art. For example, radioactive isotopes can bedetected by scintillation counting or direct visualization; fluorescentmolecules can be detected with fluorescent spectrophotometers;phosphorescent molecules can be detected with a spectrophotometer ordirectly visualized with a camera; enzymes can be detected by detectionor visualization of the product of a reaction catalyzed by the enzyme;antibodies can be detected by detecting a secondary detection labelcoupled to the antibody. As used herein, detection molecules aremolecules which interact with amplified nucleic acid and to which one ormore detection labels are coupled.

J. Detection Probes

Detection probes are labeled oligonucleotides having sequencecomplementary to detection tags on amplified nucleic acids. Thecomplementary portion of a detection probe can be any length thatsupports specific and stable hybridization between the detection probeand the detection tag. For this purpose, a length of 10 to 35nucleotides is preferred, with a complementary portion of a detectionprobe 16 to 20 nucleotides long being most preferred. Detection probescan contain any of the detection labels described above. Preferredlabels are biotin and fluorescent molecules. A particularly preferreddetection probe is a molecular beacon. Molecular beacons are detectionprobes labeled with fluorescent moieties where the fluorescent moietiesfluoresce only when the detection probe is hybridized (Tyagi and Kramer,Nature Biotechnol. 14:303–309 (1995)). The use of such probes eliminatesthe need for removal of unhybridized probes prior to label detectionbecause the unhybridized detection probes will not produce a signal.This is especially useful in multiplex assays.

K. Address Probes

An address probe is an oligonucleotide having a sequence complementaryto address tags on primers. The complementary portion of an addressprobe can be any length that supports specific and stable hybridizationbetween the address probe and the address tag. For this purpose, alength of 10 to 35 nucleotides is preferred, with a complementaryportion of an address probe 12 to 18 nucleotides long being mostpreferred. An address probe can contain a single complementary portionor multiple complementary portions. Preferably, address probes arecoupled, either directly or via a spacer molecule, to a solid-statesupport. Such a combination of address probe and solid-state support area preferred form of solid-state detector.

L. Oligonucleotide Synthesis

Primers, detection probes, address probes, and any otheroligonucleotides can be synthesized using established oligonucleotidesynthesis methods. Methods to produce or synthesize oligonucleotides arewell known in the art. Such methods can range from standard enzymaticdigestion followed by nucleotide fragment isolation (see for example,Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989)Chapters 5, 6) to purely synthetic methods, for example, by thecyanoethyl phosphoramidite method. Solid phase chemical synthesis of DNAfragments is routinely performed using protected nucleoside cyanoethylphosphoramidites (S. L. Beaucage et al. (1981) Tetrahedron Lett.22:1859). In this approach, the 3′-hydroxyl group of an initial5′-protected nucleoside is first covalently attached to the polymersupport (R. C. Pless et al. (1975) Nucleic Acids Res. 2:773 (1975)).Synthesis of the oligonucleotide then proceeds by deprotection of the5′-hydroxyl group of the attached nucleoside, followed by coupling of anincoming nucleoside-3′-phosphoramidite to the deprotected hydroxyl group(M. D. Matteucci et a. (1981) J. Am. Chem. Soc. 103:3185). The resultingphosphite triester is finally oxidized to a phosphorotriester tocomplete the internucleotide bond (R. L. Letsinger et al. (1976) J. Am.Chem. Soc. 9:3655). Alternatively, the synthesis of phosphorothioatelinkages can be carried out by sulfurization of the phosphite triester.Several chemicals can be used to perform this reaction, among them3H-1,2-benzodithiole-3-one, 1,1-dioxide (R. P. Iyer, W. Egan, J. B.Regan, and S. L. Beaucage, J. Am. Chem. Soc., 1990, 112, 1253–1254). Thesteps of deprotection, coupling and oxidation are repeated until anoligonucleotide of the desired length and sequence is obtained. Othermethods exist to generate oligonucleotides such as the H-phosphonatemethod (Hall et al, (1957) J. Chem. Soc., 3291–3296) or thephosphotriester method as described by Ikuta et al., Ann. Rev. Biochem.53:323–356 (1984), (phosphotriester and phosphite-triester methods), andNarang et al., Methods Enzymol., 65:610–620 (1980), (phosphotriestermethod). Protein nucleic acid molecules can be made using known methodssuch as those described by Nielsen et al., Bioconjug. Chem. 5:3–7(1994). Other forms of oligonucleotide synthesis are described in U.S.Pat. Nos. 6,294,664 and 6,291,669.

The nucleotide sequence of an oligonucleotide is generally determined bythe sequential order in which subunits of subunit blocks are added tothe oligonucleotide chain during synthesis. Each round of addition caninvolve a different, specific nucleotide precursor, or a mixture of oneor more different nucleotide precursors. In general, degenerate orrandom positions in an oligonucleotide can be produced by using amixture of nucleotide precursors representing the range of nucleotidesthat can be present at that position. Thus, precursors for A and T canbe included in the reaction for a particular position in anoligonucleotide if that position is to be degenerate for A and T.Precursors for all four nucleotides can be included for a fullydegenerate or random position. Completely random oligonucleotides can bemade by including all four nucleotide precursors in every round ofsynthesis. Degenerate oligonucleotides can also be made having differentproportions of different nucleotides. Such oligonucleotides can be made,for example, by using different nucleotide precursors, in the desiredproportions, in the reaction.

Many of the oligonucleotides described herein are designed to becomplementary to certain portions of other oligonucleotides or nucleicacids such that stable hybrids can be formed between them. The stabilityof these hybrids can be calculated using known methods such as thosedescribed in Lesnick and Freier, Biochemistry 34:10807–10815 (1995),McGraw et al., Biotechniques 8:674–678 (1990), and Rychlik et al.,Nucleic Acids Res. 18:6409–6412 (1990).

Hexamer oligonucleotides were synthesized on a Perseptive Biosystems8909 Expedite Nucleic Acid Synthesis system using standard β-cyanoethylphosphoramidite coupling chemistry on mixed dA+dC+dG+dT synthesiscolumns (Glen Research, Sterling, Va.). The four phosphoramidites weremixed in equal proportions to randomize the bases at each position inthe oligonucleotide. Oxidation of the newly formed phosphites werecarried out using the sulfurizing reagent3H-1,2-benzothiole-3-one-1,1-idoxide (Glen Research) instead of thestandard oxidizing reagent after the first and second phosphoramiditeaddition steps. The thio-phosphitylated oligonucleotides weredeprotected using 30% ammonium hydroxide (3.0 ml) in water at 55° C. for16 hours, concentrated in an OP 120 Savant Oligo Prep deprotection unitfor 2 hours, and desalted with PD10 Sephadex columns using the protocolprovided by the manufacturer.

M. DNA Polymerases

DNA polymerases useful in multiple displacement amplification must becapable of displacing, either alone or in combination with a compatiblestrand displacement factor, a hybridized strand encountered duringreplication. Such polymerases are referred to herein as stranddisplacement DNA polymerases. It is preferred that a strand displacementDNA polymerase lack a 5′ to 3′ exonuclease activity. Strand displacementis necessary to result in synthesis of multiple copies of a targetsequence. A 5′ to 3′ exonuclease activity, if present, might result inthe destruction of a synthesized strand. It is also preferred that DNApolymerases for use in the disclosed method are highly processive. Thesuitability of a DNA polymerase for use in the disclosed method can bereadily determined by assessing its ability to carry out stranddisplacement replication. Preferred strand displacement DNA polymerasesare bacteriophage φ29 DNA polymerase (U.S. Pat. Nos. 5,198,543 and5,001,050 to Blanco et al.), Bst large fragment DNA polymerase (Exo(−)Bst; Aliotta et al., Genet. Anal. (Netherlands) 12:185–195 (1996)) andexo(−)Bca DNA polymerase (Walker and Linn, Clinical Chemistry42:1604–1608 (1996)). Other useful polymerases include phage M2 DNApolymerase (Matsumoto et al., Gene 84:247 (1989)), phage φPRD1 DNApolymerase (Jung et al., Proc. Natl. Acad. Sci. USA 84:8287 (1987)),exo(−)VENT® DNA polymerase (Kong et al., J. Biol. Chem. 268:1965–1975(1993)), Klenow fragment of DNA polymerase I (Jacobsen et al., Eur. J.Biochem. 45:623–627 (1974)), T5 DNA polymerase (Chatterjee et al., Gene97:13–19 (1991)), Sequenase (U.S. Biochemicals), PRD1 DNA polymerase(Zhu and Ito, Biochim. Biophys. Acta. 1219:267–276 (1994)), and T4 DNApolymerase holoenzyme (Kaboord and Benkovic, Curr. Biol. 5:149–157(1995)). φ29 DNA polymerase is most preferred.

Strand displacement can be facilitated through the use of a stranddisplacement factor, such as helicase. It is considered that any DNApolymerase that can perform strand displacement replication in thepresence of a strand displacement factor is suitable for use in thedisclosed method, even if the DNA polymerase does not perform stranddisplacement replication in the absence of such a factor. Stranddisplacement factors useful in strand displacement replication includeBMRF1 polymerase accessory subunit (Tsurumi et al., J. Virology67(12):7648–7653 (1993)), adenovirus DNA-binding protein (Zijderveld andvan der Vliet, J. Virology 68(2):1158–1164 (1994)), herpes simplex viralprotein ICP8 (Bochmer and Lehman, J. Virology 67(2):711–715 (1993);Skaliter and Lehman, Proc. Natl. Acad. Sci. USA 91(22):10665–10669(1994)); single-stranded DNA binding proteins (SSB; Rigler and Romano,J. Biol. Chem. 270:8910–8919 (1995)); phage T4 gene 32 protein(Villemain and Giedroc, Biochemistry 35:14395–14404 (1996); and calfthymus helicase (Siegel et al., J. Biol. Chem. 267:13629–13635 (1992)).

The ability of a polymerase to carry out strand displacement replicationcan be determined by using the polymerase in a strand displacementreplication assay such as those described in Examples 1 and 5. The assayin the examples can be modified as appropriate. For example, a helicasecan be used instead of SSB. Such assays should be performed at atemperature suitable for optimal activity for the enzyme being used, forexample, 32° C. for φ29 DNA polymerase, from 46° C. to 64° C. for exo(−)Bst DNA polymerase, or from about 60° C. to 70° C. for an enzyme from ahyperthermophylic organism. For assays from 60° C. to 70° C., primerlength may be increased to provide a melting temperature appropriate forthe assay temperature. Another useful assay for selecting a polymeraseis the primer-block assay described in Kong et al., J. Biol. Chem.268:1965–1975 (1993). The assay consists of a primer extension assayusing an M13 ssDNA template in the presence or absence of anoligonucleotide that is hybridized upstream of the extending primer toblock its progress. Enzymes able to displace the blocking primer in thisassay are expected to be useful for the disclosed method.

N. Kits

The materials described above can be packaged together in any suitablecombination as a kit useful for performing the disclosed method. Kitcomponents in a given kit can be designed and adapted for use togetherin the disclosed method. For example, disclosed are kits for amplifyinggenomic DNA, the kit comprising a lysis solution, a stabilizationsolution, a set of primers, and a DNA polymerase. The components of sucha kit are described elsewhere herein. In some forms of the kit, thelysis solution can comprise potassium hydroxide, for example, 400 mMKOH. Some useful forms of lysis solution can comprise 400 mM KOH, 100 mMdithiothreitol, and 10 mM EDTA. In some forms of the kit, thestabilization solution can comprise Tris-HCl at pH 4.1. Some usefulforms of stabilization solution can comprise 800 mM Tris-HCl, pH 4.1. Insome forms of the kit, the set of primers can comprise random hexamerprimers. In some forms of the kit, the DNA polymerase can be φ29 DNApolymerase. In some forms of the kit, the kit can further comprisedeoxynucleotide triphosphates. In some forms of the kit, the kit canfurther comprise one or more detection probes. Detection probes aredescribed elsewhere herein. In some forms of the kit, the detectionprobes can each comprise a complementary portion, where thecomplementary portion is complementary to a nucleic acid sequence ofinterest. In some forms of the kit, the kit can further comprisedenaturing solution. In some forms of the kit, the kit can furthercomprise reaction mix.

Some useful kits can comprise a lysis solution, a stabilizationsolution, a set of primers, a φ29 DNA polymerase, 1M dithiotheitol, 1×Phosphase-Buffered Saline, pH 7.5, and control DNA template; where thelysis solution comprises 400 mM KOH and 10 mM EDTA, the stabilizationsolution comprises 800 mM Tris-HCl, pH 4, and the set of primerscomprises a reaction mix; where the reaction mix comprises 150 mMTris-HCl, 200 mM KCl, 40 mM MgCl₂, 20 mM (NH₄)₂SO₄, 4 mM deoxynucleotidetriphosphates, and 0.2 mM random hexamer primers.

Any of the components that can be present in a kit that can be usedtogether can be combined in a single component of the kit. Thus, areaction mix can include, for example, buffers, deoxynucleotidetriphosphates and primers. Similarly, components and solutions can bedivided into constituent parts or sub-solutions. The kits can be usedfor any purpose, generally for nucleic acid amplification. In someforms, the kit can be designed to detect nucleic acid sequences ofinterest in a genome or other nucleic acid sample. In some forms, thekit can be designed to assess a disease, condition or predisposition ofan individual based on a nucleic acid sequences of interest.

O. Mixtures

Disclosed are mixtures formed by performing, or formed during the courseof performing, any form of the disclosed method. For example, disclosedare mixtures comprising, for example, cells and lysis solution; celllysate and stabilization solution; stabilized cell lysate and one ormore primers; stabilized cell lysate and DNA polymerase; stabilized celllysate, one or more primers, and DNA polymerase; stabilized cell lysateand replicated strands; stabilized cell lysate, one or more primers, andreplicated strands; stabilized cell lysate, DNA polymerase, andreplicated strands; stabilized cell lysate, one or more primers, DNApolymerase, and replicated strands; stabilized cell lysate and one ormore detection probes; stabilized cell lysate, one or more primers, oneor more detection probes, and replicated strands; stabilized celllysate, DNA polymerase, one or more detection probes, and replicatedstrands; and stabilized cell lysate, one or more primers, DNApolymerase, one or more detection probes, and replicated strands.

Whenever the method involves mixing or bringing into contact, forexample, compositions or-components or reagents, performing the methodcreates a number of different mixtures. For example, if the methodincludes three mixing steps, after each one of these steps a uniquemixture is formed if the steps are performed sequentially. In addition,a mixture is formed at the completion of all of the steps regardless ofhow the steps were performed. The present disclosure contemplates thesemixtures, obtained by the performance of the disclosed method as well asmixtures containing any disclosed reagent, composition, or component,for example, disclosed herein.

Uses

The disclosed method and compositions are applicable to numerous areasincluding, but not limited to, analysis of nucleic acids present incells (for example, analysis of genomic DNA in cells), diseasedetection, mutation detection, gene discovery, gene mapping (molecularhaplotyping), and agricultural research. Particularly useful is wholegenome amplification. Other uses include, for example, detection ofnucleic acids in cells and on genomic DNA arrays; molecular haplotyping;mutation detection; detection of inherited diseases such as cysticfibrosis, muscular dystrophy, diabetes, hemophilia, sickle cell anemia;assessment of predisposition for cancers such as prostate cancer, breastcancer, lung cancer, colon cancer, ovarian cancer, testicular cancer,pancreatic cancer.

Method

The disclosed method is based on strand displacement replication of thenucleic acid sequences by multiple primers. The method can be used toamplify one or more specific sequences (multiple strand displacementamplification), an entire genome or other DNA of high complexity (wholegenome strand displacement amplification), or concatenated DNA (multiplestrand displacement amplification of concatenated DNA). The disclosedmethod generally involves hybridization of primers to a target nucleicacid sequence and replication of the target sequence primed by thehybridized primers such that replication of the target sequence resultsin replicated strands complementary to the target sequence. Duringreplication, the growing replicated strands displace other replicatedstrands from the target sequence (or from another replicated strand) viastrand displacement replication. As used herein, a replicated strand isa nucleic acid strand resulting from elongation of a primer hybridizedto a target sequence or to another replicated strand. Stranddisplacement replication refers to DNA replication where a growing endof a replicated strand encounters and displaces another strand from thetemplate strand (or from another replicated strand). Displacement ofreplicated strands by other replicated strands is a hallmark of thedisclosed method which allows multiple copies of a target sequence to bemade in a single, isothermic reaction.

Nucleic acids for amplification are often obtained from cellularsamples. This generally requires disruption of the cell (to make thenucleic acid accessible) and purification of the nucleic acids prior toamplification. It also generally requires the inactivation of proteinfactors such as nucleases that could degrade the DNA, or of factors suchas histones that could bind to DNA strands and impede their use as atemplate for DNA synthesis by a polymerase. There are a variety oftechniques used to break open cells, such as sonication, enzymaticdigestion of cell walls, heating, and exposure to lytic conditions.Lytic conditions typically involve use of non-physiological pH and/orsolvents. Many lytic techniques can result in damage to nucleic acids incells, including, for example, breakage of genomic DNA. In particular,use of heating to lyse cells can damage genomic DNA and reduce theamount and quality of amplification products of genomic DNA. It has beendiscovered that alkaline lysis can cause less damage to genomic DNA andcan thus result in higher quality amplification results. Alkaline lysisalso inactivates protein factors such as nucleases, histones, or otherfactors that could impede the amplification of DNA within the sample. Inaddition, it is a useful property of alkaline lysis that reducing the pHdoes not reactivate the protein factors, but that such protein factorsremain inactivated when the pH of the solution is brought back within aneutral range.

In some forms of the disclosed method, a genomic sample is prepared byexposing cells to alkaline conditions, thereby lysing the cells andresulting in a cell lysate; reducing the pH of the cell lysate to makethe pH of the cell lysate compatible with DNA replication; andincubating the cell lysate under conditions that promote replication ofthe genome of the cells by multiple displacement amplification. Alkalineconditions are conditions where the pH is greater than 9.0. Particularlyuseful alkaline conditions for the disclosed method are conditions wherethe pH is greater than 10.0. The alkaline conditions can be, forexample, those that cause a substantial number of cells to lyse, thosethat cause a significant number of cells to lyse, or those that cause asufficient number of cells to lyse. The number of lysed cells can beconsidered sufficient if the genome can be sufficiently amplified in thedisclosed method. The amplification is sufficient if enoughamplification product is produced to permit some use of theamplification product, such as detection of sequences or other analysis.The reduction in pH is generally into the neutral range of pH 9.0 to pH6.0.

The cells can be exposed to alkaline conditions by mixing the cells witha lysis solution. The amount of lysis solution mixed with the cells canbe that amount that causes a substantial number of cells to lyse orthose that cause a sufficient number of cells to lyse. Generally, thisvolume will be a function of the pH of the cell/lysis solution mixture.Thus, the amount of lysis solution to mix with cells can be determinedgenerally from the volume of the cells and the alkaline concentration ofthe lysis buffer. For example, a smaller volume of a lysis solution witha stronger base and/or higher concentration of base would be needed tocreate sufficient alkaline conditions than the volume needed of a lysissolution with a weaker base and/or lower concentration of base. Thelysis solution can be formulated such that the cells are mixed with anequal volume of the lysis solution (to produce the desired alkalineconditions).

In some embodiments, the lysis solution can comprises a base, such as anaqueous base. Useful bases include potassium hydroxide, sodiumhydroxide, potassium acetate, sodium acetate, ammonium hydroxide,lithium hydroxide, calcium hydroxide, magnesium hydroxide, sodiumcarbonate, sodium bicarbonate, calcium carbonate, ammonia, aniline,benzylamine, n-butylamine, diethylamine, dimethylamine, diphenylamine,ethylamine, ethylenediamine, methylamine, N-methylaniline, morpholine,pyridine, triethylamine, trimethylamine, aluminum hydroxide, rubidiumhydroxide, cesium hydroxide, strontium hydroxide, barium hydroxide, andDBU (1,8-diazobicyclo[5,4,0]undec-7-ene). Useful formulations of lysissolution include lysis solution comprising 400 mM KOH, lysis solutioncomprising 400 mM KOH, 100 mM dithiothreitol, and 10 mM EDTA, and lysissolution consisting of 400 mM KOH, 100 mM dithiothreitol, and 10 mMEDTA.

In some embodiments, the lysis solution can comprise a plurality ofbasic agents. As used herein, a basic agent is a compound, compositionor solution that results in alkaline conditions. In some embodiments,the lysis solution can comprise a buffer. Useful buffers includephosphate buffers, “Good” buffers (such as BES, BICINE, CAPS, EPPS,HEPES, MES, MOPS, PIPES, TAPS, TES, and TRICINE), sodium cacodylate,sodium citrate, triethylammonium acetate, triethylammonium bicarbonate,Tris, Bis-tris, and Bis-tris propane. The lysis solution can comprise aplurality of buffering agents. As used herein, a buffering agent is acompound, composition or solution that acts as a buffer. An alkalinebuffering agent is a buffering agent that results in alkalineconditions. In some embodiments, the lysis solution can comprise acombination of one or more bases, basic agents, buffers and bufferingagents.

The pH of the cell lysate can be reduced to form a stabilized celllysate. A stabilized cell lysate is a cell lysate the pH of which is inthe neutral range (from about pH 6.0 to about pH 9.0). Useful stabilizedcell lysates have a pH that allows replication of nucleic acids in thecell lysate. For example, the pH of the stabilized cell lysate isusefully at a pH at which the DNA polymerase can function. The pH of thecell lysate can be reduced by mixing the cell lysate with astabilization solution. The stabilization solution comprises a solutionthat can reduce the pH of a cell lysate exposed to alkaline conditionsas described elsewhere herein.

The amount of stabilization solution mixed with the cell lysate can bethat amount that causes a reduction in pH to the neutral range (or otherdesired pH value). Generally, this volume will be a function of the pHof the cell lysate/stabilization solution mixture. Thus, the amount ofstabilization solution to mix with the cell lysate can be determinedgenerally from the volume of the cell lysate, its pH and bufferingcapacity, and the acidic concentration of the stabilization buffer. Forexample, a smaller volume of a stabilization solution with a strongeracid and/or higher concentration of acid would be needed to reduce thepH sufficiently than the volume needed of a stabilization solution witha weaker acid and/or lower concentration of acid. The stabilizationsolution can be formulated such that the cell lysate is mixed with anequal volume of the stabilization solution (to produce the desired pH).

In some embodiments, the stabilization solution can comprise an acid.Useful acids include hydrochloric acid, sulfuric acid, phosphoric acid,acetic acid, acetylsalicylic acid, ascorbic acid, carbonic acid, citricacid, formic acid, nitric acid, perchloric acid, HF, HBr, HI, H₂S, HCN,HSCN, HClO, monochloroacetic acid, dichloroacetic acid, trichloroaceticacid, and any carboxylic acid (ethanoic, propanoic, butanoic, etc.,including both linear or branched chain carboxylic acids). In someembodiments, the stabilization solution can comprise a buffer. Usefulbuffers include Tris-HCl, HEPES, “Good” buffers (such as BES, BICINE,CAPS, EPPS, HEPES, MES, MOPS, PIPES, TAPS, TES, and TRICINE), sodiumcacodylate, sodium citrate, triethylammonium acetate, triethylammoniumbicarbonate, Tris, Bis-tris, and Bis-tris propane. Useful formulationsof stabilization solutions include stabilization solution comprising 800mM Tris-HCl; stabilization solution comprising 800 mM Tris-HCl at pH4.1; and stabilization solution consisting of 800 mM Tris-HCl, pH 4.1.

In some embodiments, the stabilization solution can comprise a pluralityof acidic agents. As used herein, an acidic agent is a compound,composition or solution that forms an acid in solution. In someembodiments, the stabilization solution can comprise a plurality ofbuffering agents. An acidic buffering agent is a buffering agent thatforms an acid in solution. In some embodiments, the stabilizationsolution can comprise a combination of one or more acids, acidic agents,buffers and buffering agents.

In some embodiments, the pH of the cell lysate can be reduced to aboutpH 9.0 or below, to about pH 8.5 or below, to about pH 8.0 or below, toabout pH 7.5 or below, to about pH 7.2 or below, or to about pH 7.0 orbelow. In some embodiments, the pH of the cell lysate can be reduced tothe range of about pH 9.0 to about pH 6.0, to the range of about pH 9.0to about pH 6.5, to the range of about pH 9.0 to about pH 6.8, to therange of about pH 9.0 to about pH 7.0, to the range of about pH 9.0 toabout pH 7.2, to the range of about pH 9.0 to about pH 7.5, to the rangeof about pH 9.0 to about pH 8.0, to the range of about pH 9.0 to aboutpH 8.5, to the range of about pH 8.5 to about pH 6.0, to the range ofabout pH 8.5 to about pH 6.5, to the range of about pH 8.5 to about pH6.8, to the range of about pH 8.5 to about pH 7.0, to the range of aboutpH 8.5 to about pH 7.2, to the range of about pH 8.5 to about pH 7.5, tothe range of about pH 8.5 to about pH 8.0, to the range of about pH 8.0to about pH 6.0, to the range of about pH 8.0 to about pH 6.5, to therange of about pH 8.0 to about pH 6.8, to the range of about pH 8.0 toabout pH 7.0, to the range of about pH 8.0 to about pH 7.2, to the rangeof about pH 8.0 to about pH 7.5, to the range of about pH 7.5 to aboutpH 6.0, to the range of about pH 7.5 to about pH 6.5, to the range ofabout pH 7.5 to about pH 6.8, to the range of about pH 7.5 to about pH7.0, to the range of about pH 7.5 to about pH 7.2, to the range of aboutpH 7.2 to about pH 6.0, to the range of about pH 7.2 to about pH 6.5, tothe range of about pH 7.2 to about pH 6.8, to the range of about pH 7.2to about pH 7.0, to the range of about pH 7.0 to about pH 6.0, to therange of about pH 7.0 to about pH 6.5, to the range of about pH 7.0 toabout pH 6.8, to the range of about pH 6.8 to about pH 6.0, to the rangeof about pH 6.8 to about pH 6.5, or to the range of about pH 6.5 toabout pH 6.0. In some embodiments, the pH of the cell lysate can bereduced to any range having any combination of endpoints from about pH6.0 to about pH 9.0 All such endpoints and ranges are specifically andseparately contemplated.

In some embodiments, the cells are not lysed by heat. Those of skill inthe art will understand that different cells under different conditionswill be lysed at different temperatures and so can determinetemperatures and times at which the cells will not be lysed by heat. Ingeneral, the cells are not subjected to heating above a temperature andfor a time that would cause substantial cell lysis in the absence of thealkaline conditions used. As used herein, substantial cell lysis refersto lysis of 90% or greater of the cells exposed to the alkalineconditions. Significant cell lysis refers to lysis of 50% or more of thecells exposed to the alkaline conditions. Sufficient cell lysis refersto lysis of enough of the cells exposed to the alkaline conditions toallow synthesis of a detectable amount of amplification products bymultiple strand displacement amplification. In general, the alkalineconditions used in the disclosed method need only cause sufficient celllysis. It should be understood that alkaline conditions that could causesignificant or substantial cell lysis need not result in significant orsubstantial cell lysis when the method is performed.

In some embodiments, the cells are not subjected to heatingsubstantially or significantly above the temperature at which the cellsgrow. As used herein, the temperature at which the cells grow refers tothe standard temperature, or highest of different standard temperatures,at which cells of the type involved are cultured. In the case of animalcells, the temperature at which the cells grow refers to the bodytemperature of the animal. In other embodiments, the cells are notsubjected to heating substantially or significantly above thetemperature of the amplification reaction (where the genome isreplicated).

In some embodiments, the cell lysate is not subjected to purificationprior to the amplification reaction. In the context of the disclosedmethod, purification generally refers to the separation of nucleic acidsfrom other material in the cell lysate. It has been discovered thatmultiple displacement amplification can be performed on unpurified andpartially purified samples. It is commonly thought that amplificationreactions cannot be efficiently performed using unpurified nucleic acid.In particular, PCR is very sensitive to contaminants.

Forms of purification include centrifugation, extraction,chromatography, precipitation, filtration, and dialysis. Partiallypurified cell lysate includes cell lysates subjected to centrifugation,extraction, chromatography, precipitation, filtration, and dialysis.Partially purified cell lysate generally does not include cell lysatessubjected to nucleic acid precipitation or dialysis. As used herein,separation of nucleic acid from other material refers to physicalseparation such that the nucleic acid to be amplified is in a differentcontainer or container from the material. Purification does not requireseparation of all nucleic acid from all other materials. Rather, what isrequired is separation of some nucleic acid from some other material. Asused herein in the context of nucleic acids to be amplified,purification refers to separation of nucleic acid from other material.In the context of cell lysates, purification refers to separation ofnucleic acid from other material in the cell lysate. As used herein,partial purification refers to separation of nucleic acid from some, butnot all, of other material with which the nucleic acid is mixed. In thecontext of cell lysates, partial purification refers to separation ofnucleic acid from some, but not all, of the other material in the celllysate.

Unless the context clearly indicates otherwise, reference herein to alack of purification, lack of one or more types of purification orseparation operations or techniques, or exclusion of purification or oneor more types of purification or separation operations or techniquesdoes not encompass the exposure of cells to alkaline conditions (or theresults thereof) the reduction of pH of a cell lysate (or the resultsthereof). That is, to the extent that the alkaline conditions and pHreduction of the disclosed method produce an effect that could beconsidered “purification” or “separation,” such effects are excludedfrom the definition of purification and separation when those terms areused in the context of processing and manipulation of cell lysates andstabilized cell lysates (unless the context clearly indicatesotherwise).

As used herein, substantial purification refers to separation of nucleicacid from at least a substantial portion of other material with whichthe nucleic acid is mixed. In the context of cell lysates, substantialpurification refers to separation of nucleic acid from at least asubstantial portion of the other material in the cell lysate. Asubstantial portion refers to 90% of the other material involved.Specific levels of purification can be referred to as a percentpurification (such as 95% purification and 70% purification). A percentpurification refers to purification that results in separation fromnucleic acid of at least the designated percent of other material withwhich the nucleic acid is mixed.

Denaturation of nucleic acid molecules to be amplified is common inamplification techniques. This is especially true when amplifyinggenomic DNA. In particular, PCR uses multiple denaturation cycles.Denaturation is generally used to make nucleic acid strands accessibleto primers. It was discovered that the target nucleic acids, genomicDNA, for example, need not be denatured for efficient multipledisplacement amplification. It was also discovered that elimination of adenaturation step and denaturation conditions has additional advantagessuch as reducing sequence bias in the amplified products. In someembodiments, the nucleic acids in the cell lysate are not denatured byheating. In some embodiments, the cell lysate is not subjected toheating substantially or significantly above the temperature at whichthe cells grow. In other embodiments, the cell lysate is not subjectedto heating substantially or significantly above the temperature of theamplification reaction (where the genome is replicated). The disclosedmultiple displacement amplification reaction is generally conducted at asubstantially constant temperature (that is, the amplification reactionis substantially isothermic), and this temperature is generally belowthe temperature at which the nucleic acids would be notably denatured.As used herein, notable denaturation refers to denaturation of 10% orgreater of the base pairs.

In preferred forms of the disclosed method, the nucleic acid sample ortemplate nucleic acid is not subjected to denaturing conditions and/orno denaturation step is used. In some forms of the disclosed method, thenucleic acid sample or template nucleic acid is not subjected to heatdenaturing conditions and/or no heat denaturation step is used. Itshould be understood that while sample preparation (for example, celllysis and processing of cell extracts) may involve conditions that mightbe considered denaturing (for example, treatment with alkali), thedenaturation conditions or step eliminated in some forms of thedisclosed method refers to denaturation steps or conditions intended andused to make nucleic acid strands accessible to primers. Suchdenaturation is commonly a heat denaturation, but can also be otherforms of denaturation such as chemical denaturation. It should beunderstood that in the disclosed method where the nucleic acid sample ortemplate nucleic acid is not subjected to denaturing conditions, thetemplate strands are accessible to the primers (since amplificationoccurs). However, the template stands are not made accessible viageneral denaturation of the sample or template nucleic acids.

The efficiency of a DNA amplification procedure may be described forindividual loci as the percent representation, where the percentrepresentation is 100% for a locus in genomic DNA as purified fromcells. For 10,000-fold amplification, the average representationfrequency was 141% for 8 loci in DNA amplified without heat denaturationof the template, and 37% for the 8 loci in DNA amplified with heatdenaturation of the template. The omission of a heat denaturation stepresults in a 3.8-fold increase in the representation frequency foramplified loci. Amplification bias may be calculated between two samplesof amplified DNA or between a sample of amplified DNA and the templateDNA it was amplified from. The bias is the ratio between the values forpercent representation for a particular locus. The maximum bias is theratio of the most highly represented locus to the least representedlocus. For 10,000-fold amplification, the maximum amplification bias was2.8 for DNA amplified without heat denaturation of the template, and50.7 for DNA amplified with heat denaturation of the template. Theomission of a heat denaturation step results in an 18-fold decrease inthe maximum bias for amplified loci.

In another form of the method, the primers can be hexamer primers. Itwas discovered that such short, 6 nucleotide primers can still primemultiple strand displacement replication efficiently. Such short primersare easier to produce as a complete set of primers of random sequence(random primers) than longer primers at least because there are fewer tomake. In another form of the method, the primers can each contain atleast one modified nucleotide such that the primers are nucleaseresistant. In another form of the method, the primers can each containat least one modified nucleotide such that the melting temperature ofthe primer is altered relative to a primer of the same sequence withoutthe modified nucleotide(s). In another form of the method, the DNApolymerase can be φ29 DNA polymerase. It was discovered that φ29 DNApolymerase produces greater amplification in multiple displacementamplification. The combination of two or more of the above features alsoyields improved results in multiple displacement amplification. In apreferred embodiment, for example, the target sample is not subjected todenaturing conditions, the primers are hexamer primers and containmodified nucleotides such that the primers are nuclease resistant, andthe DNA polymerase is φ29 DNA polymerase. The above features areespecially useful in whole genome strand displacement amplification(WGSDA).

In another form of the disclosed method, the method includes labeling ofthe replicated strands (that is, the strands produced in multipledisplacement amplification) using terminal deoxynucleotidyl transferase.The replicated strands can be labeled by, for example, the addition ofmodified nucleotides, such as biotinylated nucleotides, fluorescentnucleotides, 5 methyl dCTP, BrdUTP, or 5-(3-aminoallyl)-2′-deoxyuridine5′-triphosphates, to the 3′ ends of the replicated strands.

Some forms of the disclosed method provide amplified DNA of higherquality relative to previous methods due to the lack of a heatdenaturation treatment of the DNA that is the target for amplification.Thus, the template DNA does not undergo the strand breakage eventscaused by heat treatment and the amplification that is accomplished by asingle DNA polymerase extends farther along template strands ofincreased length.

In one form of the disclosed method, a small amount of purifieddouble-strand human genomic DNA (1 ng, for example) can be mixed withexonuclease-resistant random hexamer primers and φ29 DNA polymeraseunder conditions that favor DNA synthesis. For example, the mixture cansimply be incubated at 30° C. and multiple displacement amplificationwill take place. Thus, any single-stranded or duplex DNA may be used,without any additional treatment, making the disclosed method a simple,one-step procedure. Since so little DNA template is required, a majoradvantage of the disclosed method is that DNA template may be taken frompreparations that contain levels of contaminants that would inhibitother DNA amplification procedures such as PCR. For MDA the sample maybe diluted so that the contaminants fall below the concentration atwhich they would interfere with the reaction. The disclosed method canbe performed (and the above advantages achieved) using any type ofsample, including, for example, bodily fluids such as urine, semen,lymphatic fluid, cerebrospinal fluid, and amniotic fluid.

The need for only small amounts of DNA template in the disclosed methodmeans that the method is useful for DNA amplification from very smallsamples. In particular, the disclosed method may be used to amplify DNAfrom a single cell. The ability to obtain analyzable amounts of nucleicacid from a single cell (or similarly small sample) has manyapplications in preparative, analytical, and diagnostic procedures suchas prenatal diagnostics. Other examples of biological samples containingonly small amounts of DNA for which amplification by the disclosedmethod would be useful are material excised from tumors or otherarchived medical samples, needle aspiration biopsies, clinical samplesarising from infections, such as nosocomial infections, forensicsamples, or museum specimens of extinct species.

More broadly, the disclosed method is useful for applications in whichthe amounts of DNA needed are greater than the supply. For example,procedures that analyze DNA by chip hybridization techniques are limitedby the amounts of DNA that can be purified from typically sized bloodsamples. As a result many chip hybridization procedures utilize PCR togenerate a sufficient supply of material for the high-throughputprocedures. The disclosed method presents a useful technique for thegeneration of plentiful amounts of amplified DNA that faithfullyreproduces the locus representation frequencies of the startingmaterial.

Whole genome amplification by MDA can be carried out directly from bloodor cells bypassing the need to isolate pure DNA. For example, blood orother cells can be lysed by dilution with an equal volume of 2× AlkalineLysis Buffer (400 mM KOH, 100 mM dithiothreitol, and 10 mM EDTA), anexample of a lysis solution, and incubated 10 minutes on ice. The lysedcells can be neutralized with the same volume of Neutralization Buffer(800 mM Tris-HCl, pH 4.1), an example of a stabilization solution.Preparations of lysed blood or cells (for example, 1 ml) can useddirectly as template in MDA reactions (for example, 100 ml). If desired,prior to lysis, blood can be diluted 3-fold in phosphate buffered saline(PBS) and tissue culture cells can be diluted to 30,000 cells/ml in PBS.

A specific embodiment of the disclosed method is described in Example 1,wherein whole genome amplification is performed by MDA without heattreatment of the human template DNA. As shown in the example, thedisclosed method produces a DNA amplification product with improvedperformance in genetic assays compared to amplification performed withheat treatment of the template DNA. The longer DNA products producedwithout heat treatment of the template yield larger DNA fragments inSouthern blotting and genetic analysis using RFLP.

The breakage of DNA strands by heat treatment is demonstrated directlyin Example 2, while the decreased rate and yield of DNA amplificationfrom heat-treated DNA is depicted in Example 3. The decrease in DNAproduct strand length resulting from heat treatment of the DNA templateis demonstrated in Example 4.

A specific form of the disclosed method is described in Example 5,wherein purified human genomic DNA is amplified by MDA without heattreatment of the template. As shown in the example, the disclosed methodproduces for a DNA amplification product with no loss of locusrepresentation when used as a substrate in quantitative PCR assayscompared to DNA amplified with heat treatment of the template.

Another specific form of the disclosed method is described in Example 6,wherein purified human genomic DNA is amplified by MDA without heattreatment of the template. As shown in the example, the disclosed methodproduces a DNA amplification product with a low amplification bias, withthe variation in representation among eight different loci varying byless than 3.0. In contrast, the amplification bias of DNA productsamplified by two PCR-based amplification methods, PEP and DOP-PCR,varies between two and six orders of magnitude.

Another specific form of the disclosed method is described in Example 7,wherein the amplification of c-jun sequences using specific, nestedprimers from a human genomic DNA template is enhanced by omission of aDNA template heat denaturation step.

Another specific form of the disclosed method is described in Example 8,wherein human genomic DNA is amplified in the absence of a heattreatment step directly from whole blood or from tissue culture cellswith the same efficiency as from purified DNA. The DNA amplifieddirectly from blood or cells has substantially the same locusrepresentation values as DNA amplified from purified human DNA template.This represents an advantage over other amplification procedures such asPCR, since components such as heme in whole blood inhibit PCR andnecessitate a purification step before DNA from blood can be used as aPCR template.

Another specific form of the disclosed method is described in Example 9,wherein purified human genomic DNA is amplified by MDA without heattreatment of the template in the presence of 70% AA-dUTP/30% dTTP. Asshown in the example, the disclosed method provides for a DNAamplification product with the same low amplification bias as for DNAamplified in the presence of 100% dTTP.

Also disclosed is a method for amplifying and repairing damaged DNA.This method is useful, for example, for amplifying degraded genomic DNA.The method involves substantially denaturing a damaged DNA sample(generally via exposure to heat and alkaline conditions), removal orreduction of the denaturing conditions (such as by reduction of the pHand temperature of the denatured DNA sample), and replicating the DNA.The damaged DNA is repaired during replication and the average length ofDNA fragments is increased. For example, the average length of DNAfragments can be increase from, for example, 2 kb in the damaged DNAsample to, for example, 10 kb or greater for the replicated DNA. Theamplified and repaired DNA is in better condition for analysis andtesting than the damaged DNA sample. For example, this technique canprovide consistent improvements in allele representation from damagedDNA samples. This repair method can result in an overall improvement inamplification of damaged DNA by increasing the average length of theproduct, increasing the quality of the amplification products by 3-fold(by, for example, increasing the marker representation in the sample),and improving the genotyping of amplified products by lowering thefrequency of allelic dropout; all compared to the results whenamplifying damaged DNA by other methods. The replication can be multipledisplacement amplification. Denaturation of the DNA sample generally iscarried out such that the DNA is not further damaged. This method cangenerally be combined or used with any of the disclosed amplificationmethods. Another form of this method can involve substantiallydenaturing a damaged DNA sample (generally via exposure to heat andalkaline conditions), reduction of the pH of the denatured DNA sample,mixing the denatured DNA sample with an undenatured DNA sample from thesame source such that the ends of DNA in the undenatured DNA sample istransiently denatured, slowly cooling the mixture of DNA samples toallow the transiently denatured ends to anneal to the denatured DNA, andreplicating the annealed DNA.

A. Whole Genome Strand Displacement Amplification

In one form of the method, referred to as whole genome stranddisplacement amplification (WGSDA), a random or partially random set ofprimers is used to randomly prime a sample of genomic nucleic acid (oranother sample of nucleic acid of high complexity). By choosing asufficiently large set of primers of random or mostly random sequence,the primers in the set will be collectively, and randomly, complementaryto nucleic acid sequences distributed throughout nucleic acid in thesample. Amplification proceeds by replication with a processivepolymerase initiated at each primer and continuing until spontaneoustermination. A key feature of this method is the displacement ofintervening primers during replication by the polymerase. In this way,multiple overlapping copies of the entire genome can be synthesized in ashort time.

Whole genome strand displacement amplification can be performed by (a)mixing a set of random or partially random primers with a genomic sample(or other nucleic acid sample of high complexity), to produce aprimer-target sample mixture, and incubating the primer-target samplemixture under conditions that promote hybridization between the primersand the genomic DNA in the primer-target sample mixture, and (b) mixingDNA polymerase with the primer-target sample mixture, to produce apolymerase-target sample mixture, and incubating the polymerase-targetsample mixture under conditions that promote replication of the genomicDNA. Strand displacement replication is preferably accomplished by usinga strand displacing DNA polymerase or a DNA polymerase in combinationwith a compatible strand displacement factor.

The method has advantages over the polymerase chain reaction since itcan be carried out under isothermal conditions. Other advantages ofwhole genome strand displacement amplification include a higher level ofamplification than whole genome PCR, amplification is lesssequence-dependent than PCR, a lack of re-annealing artifacts or geneshuffling artifacts as can occur with PCR (since there are no cycles ofdenaturation and re-annealing), and a lower amplification bias thanPCR-based genome amplification (bias of 3-fold for WGSDA versus 20- to60-fold for PCR-based genome amplification).

Following amplification, the amplified sequences can be used for anypurpose, such as uses known and established for PCR amplified sequences.For example, amplified sequences can be detected using any of theconventional detection systems for nucleic acids such as detection offluorescent labels, enzyme-linked detection systems, antibody-mediatedlabel detection, and detection of radioactive labels. A key feature ofthe disclosed method is that amplification takes place not in cycles,but in a continuous, isothermal replication. This makes amplificationless complicated and much more consistent in output. Strand displacementallows rapid generation of multiple copies of a nucleic acid sequence orsample in a single, continuous, isothermal reaction.

It is preferred that the set of primers used for WGSDA be used atconcentrations that allow the primers to hybridize at desired intervalswithin the nucleic acid sample. For example, by using a set of primersat a concentration that allows them to hybridize, on average, every 4000to 8000 bases, DNA replication initiated at these sites will extend toand displace strands being replicated from adjacent sites. It should benoted that the primers are not expected to hybridize to the targetsequence at regular intervals. Rather, the average interval will be ageneral function of primer concentration. Primers for WGSDA can also beformed from RNA present in the sample. By degrading endogenous RNA withRNase to generate a pool of random oligomers, the random oligomers canthen be used by the polymerase for amplification of the DNA. Thiseliminates any need to add primers to the reaction. Alternatively, DNasedigestion of biological samples can generate a pool of DNA oligo primersfor RNA dependent DNA amplification.

As in multiple strand displacement amplification, displacement of anadjacent strand makes it available for hybridization to another primerand subsequent initiation of another round of replication. The intervalat which primers in the set of primers hybridize to the target sequencedetermines the level of amplification. For example, if the averageinterval is short, adjacent strands will be displaced quickly andfrequently. If the average interval is long, adjacent strands will bedisplaced only after long runs of replication.

In the disclosed method, the DNA polymerase catalyzes primer extensionand strand displacement in a processive strand displacementpolymerization reaction that proceeds as long as desired. Preferredstrand displacing DNA polymerases are bacteriophage φ29 DNA polymerase(U.S. Pat. Nos. 5,198,543 and 5,001,050 to Blanco et al.), largefragment Bst DNA polymerase (Exo(−) Bst), exo(−)Bca DNA polymerase, andSequenase. During strand displacement replication one may additionallyinclude radioactive, or modified nucleotides such as bromodeoxyuridinetriphosphate, in order to label the DNA generated in the reaction.Alternatively, one may include suitable precursors that provide abinding moiety such as biotinylated nucleotides (Langer et al., Proc.Natl. Acad. Sci. USA 78:6633 (1981)).

Genome amplification using PCR, and uses for the amplified DNA, isdescribed in Zhang et al., Proc. Natl. Acad. Sci. USA 89:5847–5851(1992), Telenius et al., Genomics 13:718–725 (1992), Cheung et al.,Proc. Natl. Acad. Sci. USA 93:14676–14679 (1996), and Kukasjaarvi etal., Genes, Chromosomes and Cancer 18:94–101 (1997). The uses of theamplified DNA described in these publications are also generallyapplicable to DNA amplified using the disclosed methods. Whole GenomeStrand Displacement Amplification, unlike PCR-based whole genomeamplification, is suitable for haplotype analysis since WGSDA yieldslonger fragments than PCR-based whole genome amplification. PCR-basedwhole genome amplification is also less suitable for haplotype analysissince each cycle in PCR creates an opportunity for priming events thatresult in the association of distant sequences (in the genome) to be puttogether in the same fragment.

B. Multiple Strand Displacement Amplification

In one preferred form of the method, referred to as multiple stranddisplacement amplification (MSDA), two sets of primers are used, a rightset and a left set. Primers in the right set of primers each have aportion complementary to nucleotide sequences flanking one side of atarget nucleotide sequence and primers in the left set of primers eachhave a portion complementary to nucleotide sequences flanking the otherside of the target nucleotide sequence. The primers in the right set arecomplementary to one strand of the nucleic acid molecule containing thetarget nucleotide sequence and the primers in the left set arecomplementary to the opposite strand. The 5′ end of primers in both setsare distal to the nucleic acid sequence of interest when the primers arehybridized to the flanking sequences in the nucleic acid molecule.Preferably, each member of each set has a portion complementary to aseparate and non-overlapping nucleotide sequence flanking the targetnucleotide sequence. Amplification proceeds by replication initiated ateach primer and continuing through the target nucleic acid sequence. Akey feature of this method is the displacement of intervening primersduring replication. Once the nucleic acid strands elongated from theright set of primers reaches the region of the nucleic acid molecule towhich the left set of primers hybridizes, and vice versa, another roundof priming and replication will take place. This allows multiple copiesof a nested set of the target nucleic acid sequence to be synthesized ina short period of time.

Multiple strand displacement amplification can be performed by (a)mixing a set of primers with a target sample, to produce a primer-targetsample mixture, and incubating the primer-target sample mixture underconditions that promote hybridization between the primers and the targetsequence in the primer-target sample mixture, and (b) mixing DNApolymerase with the primer-target sample mixture, to produce apolymerase-target sample mixture, and incubating the polymerase-targetsample mixture under conditions that promote replication of the targetsequence. Strand displacement replication is preferably accomplished byusing a strand displacing DNA polymerase or a DNA polymerase incombination with a compatible strand displacement factor.

By using a sufficient number of primers in the right and left sets, onlya few rounds of replication are required to produce hundreds ofthousands of copies of the nucleic acid sequence of interest. Forexample, it can be estimated that, using right and left primer sets of26 primers each, 200,000 copies of a 5000 nucleotide amplificationtarget can be produced in 10 minutes (representing just four rounds ofpriming and replication). It can also be estimated that, using right andleft primer sets of 26 primers each, 200,000 copies of a 47,000nucleotide amplification target can be produced in 60 minutes (againrepresenting four rounds of priming and replication). These calculationsare based on a polymerase extension rate of 50 nucleotides per second.It is emphasized that reactions are continuous and isothermal—no cyclingis required.

The disclosed method has advantages over the polymerase chain reactionsince it can be carried out under isothermal conditions. No thermalcycling is needed because the polymerase at the head of an elongatingstrand (or a compatible strand-displacement factor) will displace, andthereby make available for hybridization, the strand ahead of it. Otheradvantages of multiple strand displacement amplification include theability to amplify very long nucleic acid segments (on the order of 50kilobases) and rapid amplification of shorter segments (10 kilobases orless). Long nucleic acid segments can be amplified in the disclosedmethod since there is no cycling which could interrupt continuoussynthesis or allow the formation of artifacts due to rehybridization ofreplicated strands. In multiple strand displacement amplification,single priming events at unintended sites will not lead to artifactualamplification at these sites (since amplification at the intended sitewill quickly outstrip the single strand replication at the unintendedsite).

In another form of the method, referred to as gene specific stranddisplacement amplification (GS-MSDA), target DNA is first digested witha restriction endonuclease. The digested fragments are then ligatedend-to-end to form DNA circles. These circles can be monomers orconcatemers. Two sets of primers are used for amplification, a right setand a left set. Primers in the right set of primers each have a portioncomplementary to nucleotide sequences flanking one side of a targetnucleotide sequence and primers in the left set of primers each have aportion complementary to nucleotide sequences flanking the other side ofthe target nucleotide sequence. The primers in the right set arecomplementary to one strand of the nucleic acid molecule containing thetarget nucleotide sequence and the primers in the left set arecomplementary to the opposite strand. The primers are designed to coverall or part of the sequence needed to be amplified. Preferably, eachmember of each set has a portion complementary to a separate andnon-overlapping nucleotide sequence flanking the target nucleotidesequence. Amplification proceeds by replication initiated at each primerand continuing through the target nucleic acid sequence. In one form ofGS-MSDA, referred to as linear GS-MSDA, amplification is performed witha set of primers complementary to only one strand, thus amplifying onlyone of the strands. In another form of GS-MSDA, cDNA sequences can becircularized to form single stranded DNA circles. Amplification is thenperformed with a set of primers complementary to the single-strandedcircular cDNA.

C. Multiple Strand Displacement Amplification of Concatenated DNA

In another form of the method, referred to as multiple stranddisplacement amplification of concatenated DNA (MSDA-CD), concatenatedDNA is amplified. A preferred form of concatenated DNA is concatenatedcDNA. Concatenated DNA can be amplified using a random or partiallyrandom set of primers, as in WGSDA, or using specific primerscomplementary to specific hybridization targets in the concatenated DNA.MSDA-CD is preferred for amplification of a complex mixture or sample ofrelatively short nucleic acid samples (that is, fragments generally inthe range of 100 to 6,000 nucleotides). Messenger RNA is the mostimportant example of such a complex mixture. MSDA-CD provides a meansfor amplifying all cDNAs in a cell is equal fashion. Because theconcatenated cDNA can be amplified up to 5,000-fold, MSDA-CD will permitRNA profiling analysis based on just a few cells. To perform MSDA-CD,DNA must first be subjected to a concatenation step. If an RNA sample(such as mRNA) is to be amplified, the RNA is first converted to adouble-stranded cDNA using standard methods. The cDNA, or any other setof DNA fragments to be amplified, is then converted into a DNAconcatenate, preferably with incorporation of linkers.

D. Multiple Strand Displacement Amplification of Damaged DNA

Other forms of the disclosed method can involve amplification and repairof damaged DNA. Amplification of damaged DNA can be both difficult andprovide unreliable results. For example, amplification of degraded orfragmented DNA will produce truncated products and can result in alleledropout. Preparation of genomic DNA samples in particular can result indamage to the genomic DNA (for example, degradation and fragmentation).Damaged DNA and damaged DNA samples can be amplified and repaired in thedisclosed method of amplifying damage DNA. The method generally works byhybridizing the ends of some DNA molecules in a damaged DNA sample tocomplementary sequences in the sample. Because the DNA moleculesproviding the newly associated ends will have damage at differentlocations, priming from the annealed ends can result in replication ofmore complete fragments and can mediate repair of the damaged DNA (inthe form of less damaged or undamaged replicated strands). Replicationof the undamaged replicated strands by continued multiple displacementamplification produces less damaged or undamaged amplified nucleicacids.

The method generally involves substantially denaturing a damaged DNAsample (generally via exposure to heat and alkaline conditions), removalor reduction of the denaturing conditions (such as by reduction of thepH and temperature of the denatured DNA sample), and replicating theDNA. The damaged DNA is repaired during replication and the averagelength of DNA fragments is increased. For example, the average length ofDNA fragments can be increase from, for example, 2 kb in the damaged DNAsample to, for example, 10 kb or greater for the replicated DNA. Theamplified and repaired DNA is in better condition for analysis andtesting than the damaged DNA sample. For example, this technique canprovide consistent improvements in allele representation from damagedDNA samples. This repair method can result in an overall improvement inamplification of damaged DNA by increasing the average length of theproduct, increasing the quality of the amplification products by 3-fold(by, for example, increasing the marker representation in the sample),and improving the genotyping of amplified products by lowering thefrequency of allelic dropout; all compared to the results whenamplifying damaged DNA by other methods. The replication can be multipledisplacement amplification. Denaturation of the DNA sample generally iscarried out such that the DNA is not further damaged. This method cangenerally be combined or used with any of the disclosed amplificationmethods.

In some embodiments, the method of amplifying damaged DNA can involveexposing a damaged DNA sample to conditions that promote substantialdenaturation of damaged DNA in the damaged DNA sample, thereby forming adenatured damaged DNA sample; altering the conditions to conditions thatdo not promote substantial denaturation of damaged DNA in the damagedDNA sample to form a stabilized denatured damaged DNA sample; andincubating the annealed damaged DNA under conditions that promotereplication of the damaged DNA. The annealed ends of the damaged DNAprime replication and replication of the damaged DNA results in repairof the replicated strands. The conditions that promote substantialdenaturation of damaged DNA in the damaged DNA sample can be, forexample, raising the pH of the damaged DNA sample and heating thedamaged DNA sample. The altering conditions can be, for example,reducing the pH of the denatured damaged DNA sample and cooling thedamaged DNA sample. Raising the pH can be accomplished by exposing thedamaged DNA sample to alkaline conditions. The altering conditionsgenerally can be conditions that promote annealing of the ends of thetransiently denatured damaged DNA to the substantially denatured damagedDNA. The damaged DNA sample, the denatured damaged DNA sample, or bothcan also be exposed to ionic conditions by, for example, mixing thedamaged DNA sample or denatured damaged DNA sample with an ionicsolution or including salt(s) or other ions in the denaturing solution,the stabilization solution, or both.

In the method, the damaged DNA sample can be exposed to conditions thatpromote substantial denaturation by, for example, mixing the damaged DNAsample with a denaturing solution and by heating the damaged DNA sampleto a temperature and for a length of time that substantially denaturesthe damaged DNA in the damaged DNA sample. The temperature can be, forexample, about 25° C. to about 50° C. and the length of time can be, forexample, about 5 minutes or more. The pH of the denatured damaged DNAsample can be reduced, for example, by mixing the denatured damaged DNAsample with a stabilization solution. The damaged DNA samples can be,for example, degraded DNA fragments of genomic DNA. Replication andrepair of the damaged DNA can be accomplished by incubating the damagedDNA in the presence of a DNA polymerase, such as φ29 DNA polymerase.

The damaged DNA sample can generally be slowly cooled in order toachieve the required annealing. For example, the damaged DNA sample canbe cooled at a rate of, for example, about 0.1° C. per minute or less,about 0.2° C. per minute or less, about 0.3° C. per minute or less,about 0.4° C. per minute or less, about 0.5° C. per minute or less,about 0.6° C. per minute or less, about 0.7° C. per minute or less,about 0.8° C. per minute or less, about 0.9° C. per minute or less,about 1° C. per minute or less, about 1.0° C. per minute or less, about1.1° C. per minute or less, about 1.2° C. per minute or less, about 1.3°C. per minute or less, about 10.4° C. per minute or less, about 1.5° C.per minute or less, about 1.6° C. per minute or less, about 1.8° C. perminute or less, about 2° C. per minute or less, about 2.0° C. per minuteor less, about 2.2° C. per minute or less, about 2.4° C. per minute orless, about 2.6° C. per minute or less, about 2.8° C. per minute orless, about 3° C. per minute or less, about 3.0° C. per minute or less,about 3.5° C. per minute or less, about 4° C. per minute or less, about4.0° C. per minute or less, about 5.0° C. per minute or less, about 0.1°C. per minute, about 0.2° C. per minute, about 0.3° C. per minute, about0.4° C. per minute, about 0.5° C. per minute, about 0.6° C. per minute,about 0.7° C. per minute, about 0.8° C. per minute, about 0.9° C. perminute, about 1° C. per minute, about 1.0° C. per minute, about 1.1° C.per minute, about 1.2° C. per minute, about 1.3° C. per minute, about1.4° C. per minute, about 1.5° C. per minute, about 1.6° C. per minute,about 1.8° C. per minute, about 2° C. per minute, about 2.0° C. perminute, about 2.2° C. per minute, about 2.4° C. per minute, about 2.6°C. per minute, about 2.8° C. per minute, about 3° C. per minute, about3.0° C. per minute, about 3.5° C. per minute, about 4° C. per minute,about 4.0° C. per minute, about 5.0° C. per minute, 0.1° C. per minute,0.2° C. per minute, 0.3° C. per minute, 0.4° C. per minute, 0.5° C. perminute, 0.6° C. per minute, 0.7° C. per minute, 0.8° C. per minute, 0.9°C. per minute, 1° C. per minute, 1.0° C. per minute, 1.1° C. per minute,1.2° C. per minute, 1.3° C. per minute, 1.4° C. per minute, 1.5° C. perminute, 1.6° C. per minute, 1.8° C. per minute, 2° C. per minute, 2.0°C. per minute, 2.2° C. per minute, 2.4° C. per minute, 2.6° C. perminute, 2.8° C. per minute, 3° C. per minute, 3.0° C. per minute, 3.5°C. per minute, 4° C. per minute, 4.0° C. per minute, or 5.0° C. perminute.

The rate of cooling of the damaged DNA sample can also described interms of the percent drop in temperature. Thus, cooling a damaged DNAsample that starts at 70° C. at a rate of 1% per minute or less would becooled by 0.7° C. (or less) in the first minute and 1% (or less) of theresulting temperature in the next minute. The damaged DNA sample can becooled at a rate of, for example, about 0.1% per minute or less, about0.2% per minute or less, about 0.3% per minute or less, about 0.4% perminute or less, about 0.5% per minute or less, about 0.6% per minute orless, about 0.7% per minute or less, about 0.8% per minute or less,about 0.9% per minute or less, about 1% per minute or less, about 1.0%per minute or less, about 1.1% per minute or less, about 1.2% per minuteor less, about 1.3% per minute or less, about 1.4% per minute or less,about 1.5% per minute or less, about 1.6% per minute or less, about 1.8%per minute or less, about 2% per minute or less, about 2.0% per minuteor less, about 2.2% per minute or less, about 2.4% per minute or less,about 2.6% per minute or less, about 2.8% per minute or less, about 3%per minute or less, about 3.0% per minute or less, about 3.5% per minuteor less, about 4% per minute or less, about 4.0% per minute or less,about 5.0% per minute or less, about 0.1% per minute, about 0.2% perminute, about 0.3% per minute, about 0.4% per minute, about 0.5% perminute, about 0.6% per minute, about 0.7% per minute, about 0.8% perminute, about 0.9% per minute, about 1% per minute, about 1.0% perminute, about 1.1% per minute, about 1.2% per minute, about 1.3% perminute, about 1.4% per minute, about 1.5% per minute, about 1.6% perminute, about 1.8% per minute, about 2% per minute, about 2.0% perminute, about 2.2% per minute, about 2.4% per minute, about 2.6% perminute, about 2.8% per minute, about 3% per minute, about 3.0% perminute, about 3.5% per minute, about 4% per minute, about 4.0% perminute, about 5.0% per minute, 0.1% per minute, 0.2% per minute, 0.3%per minute, 0.4% per minute, 0.5% per minute, 0.6% per minute, 0.7% perminute, 0.8% per minute, 0.9% per minute, 1% per minute, 1.0% perminute, 1.1% per minute, 1.2% per minute, 1.3% per minute, 1.4% perminute, 1.5% per minute, 1.6% per minute, 1.8% per minute, 2% perminute, 2.0% per minute, 2.2% per minute, 2.4% per minute, 2.6% perminute, 2.8% per minute, 3% per minute, 3.0% per minute, 3.5% perminute, 4% per minute, 4.0% per minute, or 5.0% per minute.

The damaged DNA sample, the denatured damaged DNA sample, or both canalso be exposed to ionic conditions by, for example, mixing the damagedDNA sample or denatured damaged DNA sample with an ionic solution orincluding salt(s) or other ions in the denaturing solution, thestabilization solution, or both. As used herein, ionic conditions refersto a state of increased ionic strength. Thus, exposure to ionicconditions refers to exposure to a higher ionic strength than existed inthe sample or solution before exposure. This will be the result when,for example, a buffer or salt is added. A solution used to make such anaddition can be referred to as an ionic solution. The ionic solution canbe a salt solution and can comprise one or more salts or other ions. Anysuitable salt or ion can be used. The salt can be, for example,Tris-HCl, Tris-EDTA, sodium chloride, potassium chloride, magnesiumchloride, sodium acetate, potassium acetate, magnesium acetate, or acombination. The Tris-HCl can be, for example, from pH 7.0 to 8.0. Thesalt can be Tris-EDTA. The ionic solution can be diluted, for example, 2to 5 fold when mixed with the damaged DNA sample. The ionic solution canbe mixed with the denatured damaged DNA sample prior to or duringaltering of the conditions.

Ionic conditions and the composition of ionic solutions generally can becan be specified by specifying a concentration of a buffer, salt, orother ion-forming compound. A combination of compounds can be used in anionic solution or to create ionic conditions. The salt solution cancomprise, for example, about 50 mM to about 500 mM Tris and about 1 mMto about 5 mM EDTA. Ionic solutions can have a salt, buffer or ionconcentration of from about 1 mM to about 2 M, from about 1 mM to about1 M, from about 1 mM to about 900 mM, from about 1 mM to about 800 mM,from about 1 mM to about 700 mM, from about 1 mM to about 600 mM, fromabout 1 mM to about 500 mM, from about 1 mM to about 400 mM, from about1 mM to about 300 mM, from about 1 mM to about 250 mM, from about 1 mMto about 200 mM, from about 1 mM to about 150 mM, from about 1 mM toabout 100 mM, from about 1 mM to about 80 mM, from about 1 mM to about60 mM, from about 1 mM to about 50 mM, from about 1 mM to about 40 mM,from about 1 mM to about 30 mM, from about 1 mM to about 20 mM, fromabout 1 mM to about 10 mM, from about 1 mM to about 5 mM, from about 1mM to about 2 mM, from about 2 mM to about 2 M, from about 2 mM to about1 M, from about 2 mM to about 900 mM, from about 2 mM to about 800 mM,from about 2 mM to about 700 mM, from about 2 mM to about 600 mM, fromabout 2 mM to about 500 mM, from about 2 mM to about 400 mM, from about2 mM to about 300 mM, from about 2 mM to about 250 mM, from about 2 mMto about 200 mM, from about 2 mM to about 150 mM, from about 2 mM toabout 00 mM, from about 2 mM to about 80 mM, from about 2 mM to about 60mM, from about 2 mM to about 50 mM, from about 2 mM to about 40 mM, fromabout 2 mM to about 30 mM, from about 2 mM to about 20 mM, from about 2mM to about 10 mM, from about 2 mM to about 5 mM, from about 5 mM toabout 2 M, from about 5 mM to about 1 M, from about 5 mM to about 900mM, from about 5 mM to about 800 mM, from about 5 mM to about 700 mM,from about 5 mM to about 600 mM, from about 5 mM to about 500 mM, fromabout 5 mM to about 400 mM, from about 5 mM to about 300 mM, from about5 mM to about 250 mM, from about 5 mM to about 200 mM, from about 5 mMto about 150 mM, from about 5 mM to about 100 mM, from about 5 mM toabout 80 mM, from about 5 mM to about 60 mM, from about 5 mM to about 50mM, from about 5 mM to about 40 mM, from about 5 mM to about 30 mM, fromabout 5 mM to about 20 mM, from about 5 mM to about 10 mM, from about 10mM to about 2 M, from about 10 mM to about 1 M, from about 10 mM toabout 900 mM, from about 10 mM to about 800 mM, from about 10 mM toabout 700 mM, from about 10 mM to about 600 mM, from about 10 mM toabout 500 mM, from about 10 mM to about 400 mM, from about 10 mM toabout 300 mM, from about 10 mM to about 250 mM, from about 10 mM toabout 200 mM, from about 10 mM to about 150 mM, from about 10 mM toabout 100 mM, from about 10 mM to about 80 mM, from about 10 mM to about60 mM, from about 10 mM to about 50 mM, from about 10 mM to about 40 mM,from about 10 mM to about 30 mM, from about 10 mM to about 20 mM, fromabout 20 mM to about 2 M, from about 20 mM to about 1 M, from about 20mM to about 900 mM, from about 20 mM to about 800 mM, from about 20 mMto about 700 mM, from about 20 mM to about 600 mM, from about 20 mM toabout 500 mM, from about 20 mM to about 400 mM, from about 20 mM toabout 300 mM, from about 20 mM to about 250 mM, from about 20 mM toabout 200 mM, from about 20 mM to about 150 mM, from about 20 mM toabout 100 mM, from about 20 mM to about 80 mM, from about 20 mM to about60 mM, from about 20 mM to about 50 mM, from about 20 mM to about 40 mM,from about 20 mM to about 30 mM, from about 30 mM to about 2 M, fromabout 30 mM to about 1 M, from about 30 mM to about 900 mM, from about30 mM to about 800 mM, from about 30 mM to about 700 mM, from about 30mM to about 600 mM, from about 30 mM to about 500 mM, from about 30 mMto about 400 mM, from about 30 mM to about 300 mM, from about 30 mM toabout 250 mM, from about 30 mM to about 200 mM, from about 30 mM toabout 150 mM, from about 30 mM to about 100 mM, from about 30 mM toabout 80 mM, from about 30 mM to about 60 mM, from about 30 mM to about50 mM, from about 30 mM to about 40 mM, from about 40 mM to about 2 M,from about 40 mM to about 1 M, from about 40 mM to about 900 mM, fromabout 40 mM to about 800 mM, from about 40 mM to about 700 mM, fromabout 40 mM to about 600 mM, from about 40 mM to about 500 mM, fromabout 40 mM to about 400 mM, from about 40 mM to about 300 mM, fromabout 40 mM to about 250 mM, from about 40 mM to about 200 mM, fromabout 40 mM to about 150 mM, from about 40 mM to about 100 mM, fromabout 40 mM to about 80 mM, from about 40 mM to about 60 mM, from about40 mM to about 50 mM, from about 50 mM to about 2 M, from about 50 mM toabout 1 M, from about 50 mM to about 900 mM, from about 50 mM to about800 mM, from about 50 mM to about 700 mM, from about 50 mM to about 600mM, from about 50 mM to about 500 mM, from about 50 mM to about 400 mM,from about 50 mM to about 300 mM, from about 50 mM to about 250 mM, fromabout 50 mM to about 200 mM, from about 50 mM to about 150 mM, fromabout 50 mM to about 100 mM, from about 50 mM to about 80 mM, from about50 mM to about 60 mM, from about 60 mM to about 2 M, from about 60 mM toabout 1 M, from about 60 mM to about 900 mM, from about 60 mM to about800 mM, from about 60 mM to about 700 mM, from about 60 mM to about 600mM, from about 60 mM to about 500 mM, from about 60 mM to about 400 mM,from about 60 mM to about 300 mM, from about 60 mM to about 250 mM, fromabout 60 mM to about 200 mM, from about 60 mM to about 150 mM, fromabout 60 mM to about 100 mM, from about 60 mM to about 80 mM, from about80 mM to about 2 M, from about 80 mM to about 1 M, from about 80 mM toabout 900 mM, from about 80 mM to about 800 mM, from about 80 mM toabout 700 mM, from about 80 mM to about 600 mM, from about 80 mM toabout 500 mM, from about 80 mM to about 400 mM, from about 80 mM toabout 300 mM, from about 80 mM to about 250 mM, from about 80 mM toabout 200 mM, from about 80 mM to about 150 mM, from about 80 mM toabout 100 mM, from about 100 mM to about 2 M, from about 100 mM to about1 M, from about 100 mM to about 900 mM, from about 100 mM to about 800mM, from about 100 mM to about 700 mM, from about 100 mM to about 600mM, from about 00 mM to about 500 mM, from about 100 mM to about 400 mM,from about 100 mM to about 300 mM, from about 100 mM to about 250 mM,from about 100 mM to about 200 mM, from about 100 mM to about 150 mM,from about 150 mM to about 2 M, from about 150 mM to about 1 M, fromabout 150 mM to about 900 mM, from about 150 mM to about 800 mM, fromabout 150 mM to about 700 mM, from about 150 mM to about 600 mM, fromabout 150 mM to about 500 mM, from about 150 mM to about 400 mM, fromabout 150 mM to about 300 mM, from about 150 mM to about 250 mM, fromabout 150 mM to about 200 mM, from about 200 mM to about 2 M, from about200 mM to about 1 M, from about 200 mM to about 900 mM, from about 200mM to about 800 mM, from about 200 mM to about 700 mM, from about 200 mMto about 600 mM, from about 200 mM to about 500 mM, from about 200 mM toabout 400 mM, from about 200 mM to about 300 mM, from about 200 mM toabout 250 mM, from about 250 mM to about 2 M, from about 250 mM to about1 M, from about 250 mM to about 900 mM, from about 250 mM to about 800mM, from about 250 mM to about 700 mM, from about 250 mM to about 600mM, from about 250 mM to about 500 mM, from about 250 mM to about 400mM, from about 250 mM to about 300 mM, from about 300 mM to about 2 M,from about 300 mM to about 1 M, from about 300 mM to about 900 mM, fromabout 300 mM to about 800 mM, from about 300 mM to about 700 mM, fromabout 300 mM to about 600 mM, from about 300 mM to about 500 mM, fromabout 300 mM to about 400 mM, from about 400 mM to about 2 M, from about400 mM to about 1 M, from about 400 mM to about 900 mM, from about 400mM to about 800 mM, from about 400 mM to about 700 mM, from about 400 mMto about 600 mM, from about 400 mM to about 500 mM, from about 500 mM toabout 2 M, from about 500 mM to about 1 M, from about 500 mM to about900 mM, from about 500 mM to about 800 mM, from about 500 mM to about700 mM, from about 500 mM to about 600 mM, from about 600 mM to about 2M, from about 600 mM to about 1 M, from about 600 mM to about 900 mM,from about 600 mM to about 800 mM, from about 600 mM to about 700 mM,from about 700 mM to about 2 M, from about 700 mM to about 1 M, fromabout 700 mM to about 900 mM, from about 700 mM to about 800 mM, fromabout 800 mM to about 2 M, from about 800 mM to about 1 M, from about800 mM to about 900 mM, from about 900 mM to about 2 M, from about 900mM to about 1 M, from about 1 M to about 2 M, about 2 M, about 1 M,about 900 mM, about 800 mM, about 700 mM, about 600 mM, about 500 mM,about 400 mM, about 300 mM, about 250 mM, about 200 mM, about 150 mM,about 100 mM, about 80 mM, about 60 mM, about 50 mM, about 40 mM, about30 mM, about 20 mM, about 10 mM, about 9 mM, about 8, mM, about 7 mM,about 6 mM, about 5 mM, about 4 mM, about 3 mM, about 2 mM, about 1 mM,2 M, 1 M, 900 mM, 800 mM, 700 mM, 600 mM, 500 mM, 400 mM, 300 mM, 250mM, 200 mM, 150 mM, 100 mM, 80 mM, 60 mM, 50 mM, 40 mM, 30 mM, 20 mM, 10mM, 9 mM, 8 mM, 7 mM, 6 mM, 5 mM, 4 mM, 3 mM, 2 mM, or 1 mM.

The disclosed method of repairing and amplifying DNA can result in anincrease in the average length of DNA fragment in a DNA sample. Thisincrease can be referred to in any suitable terms. For example, theincrease in average fragment length can be referred to by the averagefragment length of the replicated DNA fragments, the increase in averagefragment length from the average fragment length of the damaged DNAsample, and the percent increase in average fragment length. Theincrease in average fragment length can be, for example, 5%, 10%, 15%,20%, 25%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%,140%, 150%, 160%, 180%, 200%, 220%, 240%, 260%, 280%, 300%, 350%, 400%,450%, 500%, 600%, 700%, 800%, 900%, 1,000%, 5% or more, 10% or more, 15%or more, 20% or more, 25% or more, 30% or more, 40% or more, 50% ormore, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more,110% or more, 120% or more, 130% or more, 140% or more, 150% or more,160% or more, 180% or more, 200% or more, 220% or more, 240% or more,260% or more, 280% or more, 300% or more, 350% or more, 400% or more,450% or more, 500% or more, 600% or more, 700% or more, 800% or more,900% or more, 1,000% or more, about 5%, about 10%, about 15%, about 20%,about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about80%, about 90%, about 100%, about 110%, about 120%, about 130%, about140%, about 150%, about 160%, about 180%, about 200%, about 220%, about240%, about 260%, about 280%, about 300%, about 350%, about 400%, about450%, about 500%, about 600%, about 700%, about 800%, about 900%, about1,000%, about 5% or more, about 10% or more, about 15% or more, about20% or more, about 25% or more, about 30% or more, about 40% or more,about 50% or more, about 60% or more, about 70% or more, about 80% ormore, about 90% or more, about 100% or more, about 110% or more, about120% or more, about 130% or more, about 140% or more, about 150% ormore, about 160% or more, about 180% or more, about 200% or more, about220% or more, about 240% or more, about 260% or more, about 280% ormore, about 300% or more, about 350% or more, about 400% or more, about450% or more, about 500% or more, about 600% or more, about 700% ormore, about 800% or more, about 900% or more, or about 1,000% or morerelative to the average fragment length of the damaged DNA sample beforethe method.

Following the repair method, the average fragment length can be, forexample, 2 kilobases (kb), 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, 5 kb, 5.5kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, 16kb, 18 kb, 20 kb, 22 kb, 24 kb, 26 kb, 28 kb, 30 kb, 2 kb or more, 2.5kb or more, 3 kb or more, 3.5 kb or more, 4 kb or more, 4.5 kb or more,5 kb or more, 5.5 kb or more, 6 kb or more, 7 kb or more, 8 kb or more,9 kb or more, 10 kb or more, 11 kb or more, 12 kb or more, 13 kb ormore, 14 kb or more, 15 kb or more, 16 kb or more, 18 kb or more, 20 kbor more, 22 kb or more, 24 kb or more, 26 kb or more, 28 kb or more, 30kb or more, about 2 kb, about 2.5 kb, about 3 kb, about 3.5 kb, about 4kb, about 4.5 kb, about 5 kb, about 5.5 kb, about 6 kb, about 7 kb,about 8 kb, about 9 kb, about 10 kb, about 11 kb, about 12 kb, about 13kb, about 14 kb, about 15 kb, about 16 kb, about 18 kb, about 20 kb,about 22 kb, about 24 kb, about 26 kb, about 28 kb, about 30 kb, about 2kb or more, about 2.5 kb or more, about 3 kb or more, about 3.5 kb ormore, about 4 kb or more, about 4.5 kb or more, about 5 kb or more,about 5.5 kb or more, about 6 kb or more, about 7 kb or more, about 8 kbor more, about 9 kb or more, about 10 kb or more, about 11 kb or more,about 12 kb or more, about 13 kb or more, about 14 kb or more, about 15kb or more, about 16 kb or more, about 18 kb or more, about 20 kb ormore, about 22 kb or more, about 24 kb or more, about 26 kb or more,about 28 kb or more, or about 30 kb or more.

The disclosed method of amplifying damaged DNA can be combined with thedisclosed amplification of cell lysates. Thus, for example, the damagedDNA samples can be a cell lysate, where the cell lysate is produced byexposing cells to alkaline conditions. Some forms of the method caninclude exposing cells to alkaline conditions to form a cell lysate;exposing the cell lysate to conditions that promote substantialdenaturation of damaged DNA in the cell lysate; reducing the pH of thecell lysate to form a stabilized cell lysate; cooling the stabilizedcell lysate under conditions that promote annealing of the ends of thedenatured damaged DNA; and incubating the stabilized cell lysate underconditions that promote replication of the damaged DNA. Duringreplication, the annealed ends of the damaged DNA prime replication andreplication of the damaged DNA results in repair of the replicatedstrands and an increase in the average length of DNA fragment. The celllysate can be a whole genome. Replication of the genome results inreplicated strands, where during replication at least one of thereplicated strands is displaced from the genome by strand displacementreplication of another replicated strand.

In another form, the method works by hybridizing the ends of some DNAmolecules in a sample to complementary sequences in a damaged DNAsample. Generally, the damaged DNA sample and the DNA sample providingthe annealed ends or from the same source or even the same sample.Because the DNA molecules providing the newly associated ends anddamaged DNA molecules will have damage at different locations, primingfrom the annealed ends can result in replication of more completefragments and can mediate repair of the damaged DNA (in the form of lessdamaged or undamaged replicated strands). Replication of the undamagedreplicated strands by continued multiple displacement amplificationproduces less damaged or undamaged amplified nucleic acids.

The method generally involves substantially denaturing a damaged DNAsample (generally via exposure to heat and alkaline conditions),reduction of the pH of the denatured DNA sample, mixing the denaturedDNA sample with an undenatured DNA sample from the same source such thatthe ends of DNA in the undenatured DNA sample is transiently denatured,slowly cooling the mixture of DNA samples to allow the transientlydenatured ends to anneal to the denatured DNA, and replicating theannealed DNA. The damaged DNA is repaired during replication. Thereplication can be multiple displacement amplification. Substantialdenaturation and transient denaturation of the DNA samples generally iscarried out such that the DNA is not further damaged. This method cangenerally be combined or used with any of the disclosed amplificationmethods.

In some embodiments, the method of amplifying damaged DNA can involveexposing a first damaged DNA sample to conditions that promotesubstantial denaturation of damaged DNA in the first damaged DNA sample,thereby forming a denatured damaged DNA sample; reducing the pH of thedenatured damaged DNA sample to form a stabilized denatured damaged DNAsample; mixing a second damaged DNA sample with the stabilized denatureddamaged DNA sample under conditions that promote transient denaturationof the ends of damaged DNA in the second sample and that maintainsubstantial denaturation of the damaged DNA in the stabilized denatureddamaged DNA sample, thereby forming a damaged DNA mixture; cooling thedamaged DNA mixture under conditions that promote annealing of the endsof the transiently denatured damaged DNA to the substantially denatureddamaged DNA; and incubating the annealed damaged DNA under conditionsthat promote replication of the damaged DNA. The annealed ends of thedamaged DNA prime replication and replication of the damaged DNA resultsin repair of the replicated strands.

In the method, the first damaged DNA sample can be exposed to conditionsthat promote substantial denaturation by, for example, mixing the firstdamaged DNA sample with a denaturing solution and by heating the firstdamaged DNA sample to a temperature and for a length of time thatsubstantially denatures the damaged DNA in the first damaged DNA sample.The temperature can be, for example, about 25° C. to about 50° C. andthe length of time can be, for example, about 5 minutes or more. The pHof the denatured damaged DNA sample can be reduced, for example, bymixing the denatured damaged DNA sample with a stabilization solution.The damaged DNA samples can be, for example, degraded DNA fragments ofgenomic DNA. The first and second damaged DNA samples can be from thesame source, and in particular can be a portion of the same damaged DNAsample. The second damaged DNA sample can be mixed with the stabilizeddenatured damaged DNA sample at a temperature and for a length of timethat transiently denatures the damaged DNA in the second damaged DNAsample. For example, the temperature can be about 70° C. or less and thelength of time can be about 30 seconds or less. The desired effect canalso be achieved by maintaining the mixture at the temperature to whichthe first damaged DNA sample is exposed for denaturation. Replicationand repair of the damaged DNA can be accomplished by incubating theannealed damaged DNA in the presence of a DNA polymerase, such as φ29DNA polymerase.

The disclosed method of amplifying damaged DNA can be combined with thedisclosed amplification of cell lysates. Thus, for example, the firstand second damaged DNA samples can be portions of a cell lysate, wherethe cell lysate is produced by exposing cells to alkaline conditions.The pH of the second damaged DNA sample can be reduced prior to mixingwith the stabilized denatured damaged DNA. Some forms of the method caninclude exposing cells to alkaline conditions to form a cell lysate;exposing a first portion of the cell lysate to conditions that promotesubstantial denaturation of damaged DNA in the first portion of the celllysate; reducing the pH of the first portion of the cell lysate to forma first stabilized cell lysate and reducing the pH of a second portionof the cell lysate to form a second stabilized cell lysate; mixing thesecond stabilized cell lysate with the first stabilized cell lysateunder conditions that promote transient denaturation of the ends ofdamaged DNA in the second stabilized cell lysate and that maintainsubstantial denaturation of the damaged DNA in the first stabilized celllysate, thereby forming a stabilized cell lysate mixture; cooling thestabilized cell lysate mixture under conditions that promote annealingof the ends of the transiently denatured damaged DNA to thesubstantially denatured damaged DNA; and incubating the stabilized celllysate mixture under conditions that promote replication of the damagedDNA. During replication, the annealed ends of the damaged DNA primereplication and replication of the damaged DNA results in repair of thereplicated strands. The cell lysate can be a whole genome. Replicationof the genome results in replicated strands, where during replication atleast one of the replicated strands is displaced from the genome bystrand displacement replication of another replicated strand.

E. Modifications and Additional Operations

1. Detection of Amplification Products

Amplification products can be detected directly by, for example, primarylabeling or secondary labeling, as described below.

i. Primary Labeling

Primary labeling consists of incorporating labeled moieties, such asfluorescent nucleotides, biotinylated nucleotides,digoxygenin-containing nucleotides, or bromodeoxyuridine, during stranddisplacement replication. For example, one may incorporate cyanine dyedeoxyuridine analogs (Yu et al., Nucleic Acids Res., 22:3226–3232(1994)) at a frequency of 4 analogs for every 100 nucleotides. Apreferred method for detecting nucleic acid amplified in situ is tolabel the DNA during amplification with BrdUrd, followed by binding ofthe incorporated BrdU with a biotinylated anti-BrdU antibody (ZymedLabs, San Francisco, Calif.), followed by binding of the biotin moietieswith Streptavidin-Peroxidase (Life Sciences, Inc.), and finallydevelopment of fluorescence with Fluorescein-tyramide (DuPont de Nemours& Co., Medical Products Dept.). Other methods for detecting nucleic acidamplified in situ include labeling the DNA during amplification with5-methylcytosine, followed by binding of the incorporated5-methylcytosine with an antibody (Sano et al., Biochim. Biophys. Acta951:157–165 (1988)), or labeling the DNA during amplification withaminoallyl-deoxyuridine, followed by binding of the incorporatedaminoallyl-deoxyuridine with an Oregon Green® dye (Molecular Probes,Eugene, Oreg.) (Henegariu et al., Nature Biotechnology 18:345–348(2000)).

Another method of labeling amplified nucleic acids is to incorporate5-(3-aminoallyl)-dUTP (AAdUTP) in the nucleic acid during amplificationfollowed by chemical labeling at the incorporated nucleotides.Incorporated 5-(3-aminoallyl)-deoxyuridine (AAdU) can be coupled tolabels that have reactive groups that are capable of reacting with aminegroups. AAdUTP can be prepared according to Langer et al. (1981). Proc.Natl. Acad. Sci. USA. 78: 6633–37. Other modified nucleotides can beused in analogous ways. That is, other modified nucleotides with minimalmodification can be incorporated during replication and labeled afterincorporation.

Examples of labels suitable for addition to AAdUTP are radioactiveisotopes, fluorescent molecules, phosphorescent molecules, enzymes,antibodies, and ligands. Examples of suitable fluorescent labels includefluorescein isothiocyanate (FITC), 5,6-carboxymethyl fluorescein, Texasred, nitrobenz-2-oxa-1,3-diazol-4-yl (NBD), coumarin, dansyl chloride,rhodamine, amino-methyl coumarin (AMCA), Eosin, Erythrosin, BODIPY®,Cascade Blue®, Oregon Green®, pyrene, lissamine, xanthenes, acridines,oxazines, phycoerythrin, macrocyclic chelates of lanthanide ions such asquantum dye™, fluorescent energy transfer dyes, such as thiazoleorange-ethidium heterodimer, and the cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5and Cy7. Examples of other specific fluorescent labels include3-Hydroxypyrene 5,8,10-Tri Sulfonic acid, 5-Hydroxy Tryptamine (5-HT),Acid Fuchsin, Alizarin Complexon, Alizarin Red, Allophycocyanin,Aminocoumarin, Anthroyl Stearate, Astrazon Brilliant Red 4G, AstrazonOrange R, Astrazon Red 6B, Astrazon Yellow 7 GLL, Atabrine, Auramine,Aurophosphine, Aurophosphine G, BAO 9 (Bisaminophenyloxadiazole), BCECF,Berberine Sulphate, Bisbenzamide, Blancophor FFG Solution, BlancophorSV, Bodipy Fl, Brilliant Sulphoflavin FF, Calcien Blue, Calcium Green,Calcofluor RW Solution, Calcofluor White, Calcophor White ABT Solution,Calcophor White Standard Solution, Carbostyryl, Cascade Yellow,Catecholamine, Chinacrine, Coriphosphine O, Coumarin-Phalloidin, CY3.18, CY5.1 8, CY7, Dans (1-Dimethyl Amino Naphaline 5 Sulphonic Acid),Dansa (Diamino Naphtyl Sulphonic Acid), Dansyl NH-CH3, Diamino PhenylOxydiazole (DAO), Dimethylamino-5-Sulphonic acid, DipyrrometheneboronDifluoride, Diphenyl Brilliant Flavine 7GFF, Dopamine, Erythrosin ITC,Euchrysin, FIF (Formaldehyde Induced Fluorescence), Flazo Orange, Fluo3, Fluorescamine, Fura-2, Genacryl Brilliant Red B, Genacryl BrilliantYellow 10GF, Genacryl Pink 3G, Genacryl Yellow 5GF, Gloxalic Acid,Granular Blue, Haematoporphyrin, Indo-1, Intrawhite Cf Liquid, LeucophorPAF, Leucophor SF, Leucophor WS, Lissamine Rhodamine B200 (RD200),Lucifer Yellow CH, Lucifer Yellow VS, Magdala Red, Marina Blue, MaxilonBrilliant Flavin 10 GFF, Maxilon Brilliant Flavin 8 GFF, MPS (MethylGreen Pyronine Stilbene), Mithramycin, NBD Amine, Nitrobenzoxadidole,Noradrenaline, Nuclear Fast Red, Nuclear Yellow, Nylosan BrilliantFlavin E8G, Oxadiazole, Pacific Blue, Pararosaniline (Feulgen), PhorwiteAR Solution, Phorwite BKL, Phorwite Rev, Phorwite RPA, Phosphine 3R,Phthalocyanine, Phycoerythrin R, Polyazaindacene Pontochrome Blue Black,Porphyrin, Primuline, Procion Yellow, Pyronine, Pyronine B, PyrozalBrilliant Flavin 7GF, Quinacrine Mustard, Rhodamine 123, Rhodamine 5GLD, Rhodamine 6G, Rhodamine B, Rhodamine B 200, Rhodamine B Extra,Rhodamine BB, Rhodamine BG, Rhodamine WT, Serotonin, Sevron BrilliantRed 2B, Sevron Brilliant Red 4G, Sevron Brilliant Red B, Sevron Orange,Sevron Yellow L, SITS (Primuline), SITS (Stilbene Isothiosulphonicacid), Stilbene, Snarf 1, sulpho Rhodamine B Can C, Sulpho Rhodamine GExtra, Tetracycline, Thiazine Red R, Thioflavin S, Thioflavin TCN,Thioflavin 5, Thiolyte, Thiozol Orange, Tinopol CBS, True Blue,Ultralite, Uranine B, Uvitex SFC, Xylene Orange, and XRITC.

Preferred fluorescent labels are fluorescein(5-carboxyfluorescein-N-hydroxysuccinimide ester), rhodamine(5,6-tetramethyl rhodamine), and the cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5and Cy7. The absorption and emission maxima, respectively, for thesefluors are: FITC (490 nm; 520 nm), Cy3 (554 nm; 568 nm), Cy3.5 (581 nm;588 nm), Cy5 (652 nm: 672 nm), Cy5.5 (682 nm; 703 nm) and Cy7 (755 nm;778 nm), thus allowing their simultaneous detection. Other examples offluorescein dyes include 6-carboxyfluorescein (6-FAM),2′,4′,1,4,-tetrachlorofluorescein (TET),2′,4′,5′,7′,1,4-hexachlorofluorescein (HEX), 2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyrhodamine (JOE), 2′-chloro-5′-fluoro-7′,8′-fusedphenyl-1,4-dichloro-6-carboxyfluorescein (NED), and2′-chloro-7′-phenyl-1,4-dichloro-6-carboxyfluorescein (VIC). Fluorescentlabels can be obtained from a variety of commercial sources, includingAmersham Pharmacia Biotech, Piscataway, N.J.; Molecular Probes, Eugene,Oreg.; and Research Organics, Cleveland, Ohio.

ii. Secondary Labeling with Detection Probes

Secondary labeling consists of using suitable molecular probes, referredto as detection probes, to detect the amplified nucleic acids. Forexample, a primer may be designed to contain, in its non-complementaryportion, a known arbitrary sequence, referred to as a detection tag. Asecondary hybridization step can be used to bind detection probes tothese detection tags. The detection probes may be labeled as describedabove with, for example, an enzyme, fluorescent moieties, or radioactiveisotopes. By using three detection tags per primer, and four fluorescentmoieties per each detection probe, one may obtain a total of twelvefluorescent signals for every replicated strand.

iii. Multiplexing and Hybridization Array Detection

Detection of amplified nucleic acids can be multiplexed by using sets ofdifferent primers, each set designed for amplifying different targetsequences. Only those primers that are able to find their targets willgive rise to amplified products. There are two alternatives forcapturing a given amplified nucleic acid to a fixed position in asolid-state detector. One is to include within the non-complementaryportion of the primers a unique address tag sequence for each unique setof primers. Nucleic acid amplified using a given set of primers willthen contain sequences corresponding to a specific address tag sequence.A second and preferred alternative is to use a sequence present in thetarget sequence as an address tag.

iv. Enzyme-linked Detection

Amplified nucleic acid labeled by incorporation of labeled nucleotidescan be detected with established enzyme-linked detection systems. Forexample, amplified nucleic acid labeled by incorporation of biotin usingbiotin-16-UTP (Roche Molecular Biochemicals) can be detected as follows.The nucleic acid is immobilized on a solid glass surface byhybridization with a complementary DNA oligonucleotide (address probe)complementary to the target sequence (or its complement) present in theamplified nucleic acid. After hybridization, the glass slide is washedand contacted with alkaline phosphatase-streptavidin conjugate (Tropix,Inc., Bedford, Mass.). This enzyme-streptavidin conjugate binds to thebiotin moieties on the amplified nucleic acid. The slide is again washedto remove excess enzyme conjugate and the chemiluminescent substrateCSPD (Tropix, Inc.) is added and covered with a glass cover slip. Theslide can then be imaged in a Biorad Fluorimager.

2. Linear Strand Displacement Amplification

A modified form of multiple strand displacement amplification can beperformed which results in linear amplification of a target sequence.This modified method is referred to as linear strand displacementamplification (LSDA) and is accomplished by using a set of primers whereall of the primers are complementary to the same strand of the targetsequence. In LSDA, as in MSDA, the set of primers hybridize to thetarget sequence and strand displacement amplification takes place.However, only one of the strands of the target sequence is replicated.LSDA requires thermal cycling between each round of replication to allowa new set of primers to hybridize to the target sequence. Such thermalcycling is similar to that used in PCR. Unlike linear, or single primer,PCR, however, each round of replication in LSDA results in multiplecopies of the target sequence. One copy is made for each primer used.Thus, if 20 primers are used in LSDA, 20 copies of the target sequencewill be made in each cycle of replication.

DNA amplified using MSDA and WGSDA can be further amplified bytranscription. For this purpose, promoter sequences can be included inthe non-complementary portion of primers used for strand displacementamplification, or in linker sequences used to concatenate DNA forMSDA-CD.

3. Reverse Transcription Multiple Displacement Amplification

Multiple displacement amplification can be performed on RNA or on DNAstrands reverse transcribed from RNA. A useful form of the disclosedmethod, referred to as reverse transcription multiple displacementamplification (RT-MDA) involves reverse transcribing RNA, removal of theRNA (preferably by nuclease digestion using an RNA-specific nucleasesuch as RNAse H), and multiple displacement amplification of the reversetranscribed DNA. RT-MDA can be performed using either double-strandedcDNA or using just the first cDNA strand. In the latter case, the secondcDNA strand need not be, and preferably is not, synthesized. RT-MDA isuseful for quantitative analysis of mRNA or general amplification ofmRNA sequences for any other purpose.

4. Repeat Multiple Displacement Amplification

The disclosed multiple displacement amplification operations can also besequentially combined. For example, the product of MDA can itself beamplified in another multiple displacement amplification. This isreferred to herein as repeat multiple displacement amplification (RMDA).This can be accomplished, for example, by diluting the replicatedstrands following MDA and subjecting them to a new MDA. This can berepeated one or more times. Each round of MDA will increase theamplification. Different forms of MDA, such as WGSDA and MSDA onparticular target sequences can be combined. In general, repeat MDA canbe accomplished by first bringing into contact a set of primers, DNApolymerase, and a target sample, and incubating the target sample underconditions that promote replication of the target sequence. Replicationof the target sequence results in replicated strands, wherein duringreplication at least one of the replicated strands is displaced from thetarget sequence by strand displacement replication of another replicatedstrand; and then diluting the replicated strands, bringing into contacta set of primers, DNA polymerase, and the diluted replicated strands,and incubating the replicated strands under conditions that promotereplication of the target sequence. Replication of the target sequenceresults in additional replicated strands, wherein during replication atleast one of the additional replicated strands is displaced from thetarget sequence by strand displacement replication of another additionalreplicated strand. This form of the method can be extended by performingthe following operation one or more times: diluting the additionalreplicated strands, bringing into contact a set of primers, DNApolymerase, and the diluted replicated strands, and incubating thereplicated strands under conditions that promote replication of thetarget sequence. Replication of the target sequence results inadditional replicated strands, wherein during replication at least oneof the additional replicated strands is displaced from the targetsequence by strand displacement replication of another additionalreplicated strand.

5. Using Products of Multiple Displacement Amplification

The nucleic acids produced using the disclosed method can be used forany purpose. For example, the amplified nucleic acids can be analyzed(such as by sequencing or probe hybridization) to determinecharacteristics of the amplified sequences or the presence or absence orcertain sequences. The amplified nucleic acids can also be used asreagents for assays or other methods. For example, nucleic acidsproduced in the disclosed method can be coupled or adhered to asolid-state substrate. The resulting immobilized nucleic acids can beused as probes or indexes of sequences in a sample. Nucleic acidsproduced in the disclosed method can be coupled or adhered to asolid-state substrate in any suitable way. For example, nucleic acidsgenerated by multiple strand displacement can be attached by addingmodified nucleotides to the 3′ ends of nucleic acids produced by stranddisplacement replication using terminal deoxynucleotidyl transferase,and reacting the modified nucleotides with a solid-state substrate orsupport thereby attaching the nucleic acids to the solid-state substrateor support.

Nucleic acids produced in the disclosed method also can be used asprobes or hybridization partners. For example, sequences of interest canbe amplified in the disclosed method and provide a ready source ofprobes. The replicated strands (produced in the disclosed method) can becleaved prior to use as hybridization probes. For example, thereplicated strands can be cleaved with DNAse I. The hybridization probescan be labeled as described elsewhere herein with respect to labeling ofnucleic acids produce in the disclosed method.

Nucleic acids produced in the disclosed method also can be used forsubtractive hybridization to identify sequences that are present in onlyone of a pair or set of samples. For example, amplified cDNA fromdifferent samples can be annealed and the resulting double-strandedmaterial can be separated from single-stranded material. Unhybridizedsequences would be indicative of sequences expressed in one of thesamples but not others.

Specific Embodiments

Disclosed is a method of amplifying a target nucleic acid sequence wherethe method can comprise bringing into contact a set of primers, DNApolymerase, and a target sample, and incubating the target sample underconditions that promote replication of the target sequence. Replicationof the target sequence results in replicated strands, where duringreplication at least one of the replicated strands is displaced from thetarget sequence by strand displacement replication of another replicatedstrand. The target sample is not subjected to denaturing conditions. Themethod can further comprise labeling the replicated strands usingterminal deoxynucleotidyl transferase.

The method can further comprise diluting the replicated strands,bringing into contact a set of primers, DNA polymerase, and the dilutedreplicated strands, and incubating the replicated strands underconditions that promote replication of the target sequence. Replicationof the target sequence results in additional replicated strands, whereduring replication at least one of the additional replicated strands isdisplaced from the target sequence by strand displacement replication ofanother additional replicated strand. The method can further compriseperforming the following operation one or more times: diluting theadditional replicated strands, bringing into contact a set of primers,DNA polymerase, and the diluted replicated strands, and incubating thereplicated strands under conditions that promote replication of thetarget sequence. Replication of the target sequence results inadditional replicated strands, where during replication at least one ofthe additional replicated strands is displaced from the target sequenceby strand displacement replication of another additional replicatedstrand. The method can further comprise incubating the polymerase-targetsample mixture under conditions that promote strand displacement. Themethod can further comprise bringing into contact a set of primers, DNApolymerase, and a second target sample, and incubating the second targetsample under conditions that promote replication of the target sequence.The second target sample is not subjected to denaturing conditions.Replication of the target sequence results in replicated strands, whereduring replication at least one of the replicated strands is displacedfrom the target sequence by strand displacement replication of anotherreplicated strand.

The primers can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20 nucleotides long. The primers can be 5, 6, 7, 8, 9, or 10nucleotides long. The primers can be 5, 6, 7, or 8 nucleotides long. Theprimers can be 6, 7, or 8 nucleotides long. The primers can be 6nucleotides long. The primers each can contain at least one modifiednucleotide such that the primers are resistant to 3′-5′ exonuclease. TheDNA polymerase can be bacteriophage φ29 DNA polymerase, Tts DNApolymerase, phage M2 DNA polymerase, VENT™ DNA polymerase, Klenowfragment of DNA polymerase I, T5 DNA polymerase, PRD1 DNA polymerase, T4DNA polymerase holoenzyme, T7 native polymerase T7 Sequenase®, or BstDNA polymerase. The DNA polymerase can be φ29 DNA polymerase.

The primers can be 6 nucleotides long, the primers each can contain atleast one modified nucleotide such that the primers are nucleaseresistant, and the DNA polymerase can be φ29 DNA polymerase. Thereplicated strands can be labeled by the addition of modifiednucleotides to the replicated strands. The modified nucleotides can bebiotinylated nucleotides, fluorescent nucleotides, 5 methyl dCTP,BrdUTP, or 5-(3-aminoallyl)-2′-deoxyuridine 5′-triphosphates. Modifiednucleotides can be incorporated into the replicated strands duringreplication. The modified nucleotides can be biotinylated nucleotides,fluorescent nucleotides, 5 methyl dCTP, BrdUTP, or5-(3-aminoallyl)-2′-deoxyuridine 5′-triphosphates. The modifiednucleotides can be 5-(3-aminoallyl)-2′-deoxyuridine 5′-triphosphates andthe replicated strands can be labeled by reacting labels with theincorporated 5-(3-aminoallyl)-2′-deoxyuridines. The labels can befluorescein isothiocyanate, 5,6-carboxymethyl fluorescein, Texas red,nitrobenz-2-oxa-1,3-diazol-4-yl, coumarin, dansyl chloride, rhodamine,amino-methyl coumarin, Eosin, Erythrosin, BODIPY®, Cascade Blue®, OregonGreen®, pyrene, lissamine, xanthene, acridine, oxazines, phycoerythrin,Cy3, Cy3.5, Cy5, Cy5.5, Cy7, or a combination thereof.

The target sample need not be subjected to heat denaturing conditions.The target sequence can comprise two strands, and the set of primers canhave 3 or more primers complementary to one of the strands of the targetsequence and at least one primer complementary to the other strand ofthe target sequence. The set of primers can have 3 or more primerscomplementary to the same strand of the target sequence. The set ofprimers can have 4 or more primers complementary to the same strand ofthe target sequence. The set of primers can have 4 or more primers. Theset of primers can have 5 or more primers. The conditions that promotereplication of the target sequence can be substantially isothermic. Theconditions that promote replication of the target sequence need notinvolve thermal cycling. The conditions need not include thermalcycling. The target sequence can comprise an amplification target and ahybridization target, the hybridization target can flank theamplification target, the set of primers can comprise a plurality ofprimers, each primer can comprise a complementary portion, where thecomplementary portions of the primers each can be complementary to adifferent portion of the hybridization target.

The set of primers can comprise a right set of primers and a left set ofprimers, the target sequence can be double-stranded, having a first anda second strand, the hybridization target can comprise a right and lefthybridization target, where the right hybridization target can flank theamplification target on one end and the left hybridization target canflank the amplification target on the other end. The complementaryportions of the right set primers can be (i) all complementary to thefirst strand of the target sequence and (ii) each complementary to adifferent portion of the right hybridization target, and thecomplementary portions of the left set primers can be (i) allcomplementary to the second strand of the target sequence and (ii) eachcomplementary to a different portion of the left hybridization target.

The right and left set of primers each can have 3 or more primers. Theright and left set of primers each can have 4 or more primers. The rightand left set of primers each can have 5 or more primers. The right andleft set of primers each can have the same number of primers. The targetsequence can be a nucleic acid sample of substantial complexity, and theset of primers can comprise primers having random nucleotide sequences.The target sequence can be a sample of genomic nucleic acid. The primerscan be from 5 to 20 nucleotides in length. The primers can be from 5 to10 nucleotides in length. The primers can be 6, 7, or 8 nucleotides inlength. The primers can be 6 nucleotides in length. The primers can beall of the same length. Each primer can comprise a constant portion anda random portion, where the constant portion of each primer can have thesame nucleotide sequence and the random portion of each primer can havea random nucleotide sequence.

The target sequence can be concatenated DNA. The concatenated DNA can beconcatenated with linkers. Each linker can comprise a primer complementportion, each primer can comprise a complementary portion, and thecomplementary portion of each primer can be complementary to thecomplementary portion of the linkers. The set of primers can compriseprimers having random nucleotide sequences. Each primer can comprise aconstant portion and a random portion, the constant portion of eachprimer can have the same nucleotide sequence and the random portion ofeach primer can have a random nucleotide sequence. The concatenated DNAcan be formed by ligating DNA fragments together. The DNA fragments canbe cDNA made from mRNA. The mRNA can comprise a mixture of mRNA isolatedfrom cells. The target sequence need not be a nucleic acid molecule madeup of multiple tandem repeats of a single sequence that was synthesizedby rolling circle replication.

The primers can comprise nucleotides, where one or more of thenucleotides can be ribonucleotides. From about 10% to about 50% of thenucleotides can be ribonucleotides. About 50% or more of the nucleotidescan be ribonucleotides. All of the nucleotides can be ribonucleotides.The primers can comprise nucleotides, where one or more of thenucleotides can be 2′-O-methyl ribonucleotides. From about 10% to about50% of the nucleotides can be 2′-O-methyl ribonucleotides. About 50% ormore of the nucleotides can be 2′-O-methyl ribonucleotides. All of thenucleotides can be 2′-O-methyl ribonucleotides. The primers can comprisenucleotides and the nucleotides can be a mixture of ribonucleotides and2′-O-methyl ribonucleotides. The primers can comprise nucleotides, thenucleotides can comprise bases, and one or more of the bases can beuniversal bases. At least one of the universal bases can be3-nitropyrrole. The universal base can be 5-nitroindole. From about 10%to about 50% of the bases can be universal bases. About 50% or more ofthe bases can be universal bases. All of the bases can be universalbases.

The target sample can be a biopsy sample, a blood sample, a urinesample, a cell sample, or a tissue sample. The target sample is a needleaspiration biopsy sample. Nucleic acids in the target sample need not beseparated from other material in the target sample. The target samplecan be a crude cell lysate. The target sample need not be processedbeyond cell lysis. The replicated strands can be analyzed. Thereplicated strands can be analyzed using one or more DNA chips. Thereplicated strands can be analyzed by hybridization. The replicatedstrands can be analyzed by nucleic acid sequencing. The replicatedstrands can be stored prior to, following, or both prior to andfollowing their analysis. The target sample can be a blood sample, aurine sample, a semen sample, a lymphatic fluid sample, a cerebrospinalfluid sample, amniotic fluid sample, a biopsy sample, a needleaspiration biopsy sample, a cancer sample, a tumor sample, a tissuesample, a cell sample, a cell lysate sample, a crude cell lysate sample,a forensic sample, an archeological sample, an infection sample, anosocomial infection sample, a production sample, a drug preparationsample, a biological molecule production sample, a protein preparationsample, a lipid preparation sample, a carbohydrate preparation sample,or a combination thereof.

The target sample can be a blood sample. The target sample can be aneedle aspiration biopsy sample. The target sample can be a crude celllysate sample. The target sample can be a nosocomial infection sample.The sample can be derived from a patient. The target sample can be abiological molecule production sample. Production of replicated strandscan indicate the presence of nucleic acids in the sample. The amount ofreplicated strands produced can indicate the amount of nucleic acids inthe sample. The target sample can be a drug preparation sample. Thetarget sample can be a tumor sample. The target sample can be anamniotic fluid sample. The replicated strands produced from the targetsample can represent a nucleic acid fingerprint of the sample.

The second target sample can be a sample from the same type of organismas the first target sample. The second target sample can be a samplefrom the same type of tissue as the first target sample. The secondtarget sample can be a sample from the same organism as the first targetsample. The second target sample can be obtained at a different timethan the first target sample. The second target sample can be a samplefrom a different organism than the first target sample. The secondtarget sample can be a sample from a different type of tissue than thefirst target sample. The second target sample can be a sample from adifferent species of organism than the first target sample. The secondtarget sample can be a sample from a different strain of organism thanthe first target sample. The second target sample can be a sample from adifferent cellular compartment than the first target sample.

A circular nucleic acid molecule can comprise the target sequence. Thecircular nucleic acid molecule can be produced by digesting genomic DNAwith a restriction endonuclease, and circularizing the digested DNA. Thedigested DNA can be circularized with DNA or RNA ligase. The digestedDNA can be circularized with a splint or adaptor. The target sequencecan comprise an amplification target and a hybridization target, thehybridization target can flank the amplification target, the set ofprimers can comprise a plurality of primers, each primer can comprise acomplementary portion, and the complementary portions of the primerseach can be complementary to a different portion of the hybridizationtarget.

The set of primers can comprise a right set of primers and a left set ofprimers, the target sequence can be double-stranded, having a first anda second strand, the hybridization target can comprise a right and lefthybridization target, and the right hybridization target can flank theamplification target on one end and the left hybridization target flanksthe amplification target on the other end. The complementary portions ofthe right set primers can be (i) all complementary to the first strandof the target sequence and (ii) each complementary to a differentportion of the right hybridization target, and the complementaryportions of the left set primers can be (i) all complementary to thesecond strand of the target sequence and (ii) each complementary to adifferent portion of the left hybridization target.

The circular nucleic acid molecule can be produced by circularizingcDNA. The circular nucleic acid molecule is produced by circularizingmRNA/cDNA hybrid. The mRNA/cDNA hybrid can be circularized with DNA orRNA ligase. The mRNA/cDNA hybrid can be circularized with a splint oradaptor.

Also disclosed is a method of amplifying a target nucleic acid sequencewhere the method comprises, bringing into contact a set of primers, DNApolymerase, and a target sample, and incubating the target sample underconditions that promote replication of the target sequence. Nucleicacids in the target sample are not separated from other material in thetarget sample. Replication of the target sequence results in replicatedstrands, where during replication at least one of the replicated strandsis displaced from the target sequence by strand displacement replicationof another replicated strand.

The method can further comprise bringing into contact a set of primers,DNA polymerase, and a second target sample, and incubating the secondtarget sample under conditions that promote replication of the targetsequence. The second target sample is not subjected to denaturingconditions. Replication of the target sequence results in replicatedstrands, where during replication at least one of the replicated strandsis displaced from the target sequence by strand displacement replicationof another replicated strand.

The target sample can be a crude cell lysate. The target sample need notbe processed beyond cell lysis. The target sample can be a blood sample,a urine sample, a semen sample, a lymphatic fluid sample, acerebrospinal fluid sample, amniotic fluid sample, a biopsy sample, aneedle aspiration biopsy sample, a cancer sample, a tumor sample, atissue sample, a cell sample, a cell lysate sample, a crude cell lysatesample, a forensic sample, an archeological sample, an infection sample,a nosocomial infection sample, a production sample, a drug preparationsample, a biological molecule production sample, a protein preparationsample, a lipid preparation sample, a carbohydrate preparation sample,or a combination thereof. The target sample can be a blood sample. Thetarget sample can be a needle aspiration biopsy sample. The targetsample can be a crude cell lysate sample. The target sample can be anosocomial infection sample. The sample can be derived from a patient.The target sample can be a biological molecule production sample.

Production of replicated strands can indicate the presence of nucleicacids in the sample. The amount of replicated strands produced canindicate the amount of nucleic acids in the sample. The target samplecan be a drug preparation sample. The target sample can be a tumorsample. The target sample can be an amniotic fluid sample. Thereplicated strands produced from the target sample can represent anucleic acid fingerprint of the sample.

The second target sample can be a sample from the same type of organismas the first target sample. The second target sample can be a samplefrom the same type of tissue as the first target sample. The secondtarget sample can be a sample from the same organism as the first targetsample. The second target sample can be obtained at a different timethan the first target sample. The second target sample can be a samplefrom a different organism than the first target sample. The secondtarget sample can be a sample from a different type of tissue than thefirst target sample. The second target sample can be a sample from adifferent species of organism than the first target sample. The secondtarget sample can be a sample from a different strain of organism thanthe first target sample. The second target sample can be a sample from adifferent cellular compartment than the first target sample.

Also disclosed is a method of amplifying a target nucleic acid sequence,where the method comprises, bringing into contact a set of primers, DNApolymerase, and a target sample, and incubating the target sample underconditions that promote replication of the target sequence. The targetsample is a crude cell lysate. Replication of the target sequenceresults in replicated strands, where during replication at least one ofthe replicated strands is displaced from the target sequence by stranddisplacement replication of another replicated strand.

Also disclosed is a method of amplifying a target nucleic acid sequence,where the method comprises, bringing into contact a set of primers, DNApolymerase, and a target sample, and incubating the target sample underconditions that promote replication of the target sequence. The primersare 5, 6, 7, 8, 9, or 10 nucleotides long. Replication of the targetsequence results in replicated strands, where during replication atleast one of the replicated strands is displaced from the targetsequence by strand displacement replication of another replicatedstrand.

Also disclosed is a method of amplifying a target nucleic acid sequence,where the method comprises, bringing into contact a set of primers, DNApolymerase, and a target sample, and incubating the target sample underconditions that promote replication of the target sequence. The primerseach contain at least one modified nucleotide such that the primers arenuclease resistant. Replication of the target sequence results inreplicated strands, where during replication at least one of thereplicated strands is displaced from the target sequence by stranddisplacement replication of another replicated strand.

Also disclosed is a method of amplifying a target nucleic acid sequence,where the method comprises, bringing into contact a set of primers, DNApolymerase, and a target sample, and incubating the target sample underconditions that promote replication of the target sequence. Theprimer-target sample is not subjected to denaturing conditions, theprimers are 6 nucleotides long, the primers each contain at least onemodified nucleotides such that the primers are nuclease resistant, andthe DNA polymerase is φ29 DNA polymerase. Replication of the targetsequence results in replicated strands, where during replication atleast one of the replicated strands is displaced from the targetsequence by strand displacement replication of another replicatedstrand.

Also disclosed is a method of amplifying a target nucleic acid sequence,where the method comprises, bringing into contact a set of primers, DNApolymerase, and a target sample, and incubating the target sample underconditions that promote replication of the target sequence. Replicationof the target sequence results in replicated strands, where duringreplication at least one of the replicated strands is displaced from thetarget sequence by strand displacement replication of another replicatedstrand. The method further comprises diluting the replicated strands,bringing into contact a set of primers, DNA polymerase, and the dilutedreplicated strands, and incubating the replicated strands underconditions that promote replication of the target sequence. Replicationof the target sequence results in additional replicated strands, whereduring replication at least one of the additional replicated strands isdisplaced from the target sequence by strand displacement replication ofanother additional replicated strand.

The method can further comprise performing the following operation oneor more times: diluting the additional replicated strands, bringing intocontact a set of primers, DNA polymerase, and the diluted replicatedstrands, and incubating the replicated strands under conditions thatpromote replication of the target sequence. Replication of the targetsequence results in additional replicated strands, where duringreplication at least one of the additional replicated strands isdisplaced from the target sequence by strand displacement replication ofanother additional replicated strand.

Also disclosed is a method of amplifying a target nucleic acid sequence,where the method comprises,

(a) mixing a set of primers with a target sample, to produce aprimer-target sample mixture, and incubating the primer-target samplemixture under conditions that promote hybridization between the primersand the target sequence in the primer-target sample mixture, where theprimer-target sample is not subjected to denaturing conditions, and

(b) mixing DNA polymerase with the primer-target sample mixture, toproduce a polymerase-target sample mixture, and incubating thepolymerase-target sample mixture under conditions that promotereplication of the target sequence.

The set of primers comprises a right set of primers and a left set ofprimers, the target sequence is double-stranded, having a first and asecond strand, where the right set primers are all complementary to thefirst strand of the target sequence and the left set primers are allcomplementary to the second strand of the target sequence. Replicationof the target sequence results in replicated strands, where duringreplication at least one of the replicated strands is displaced from thetarget sequence by strand displacement replication of another replicatedstrand. The right set of primers can have 4 or more primers and the leftset of primers has 4 or more primers.

Also disclosed is a method of amplifying a target nucleic acid sequence,where the method comprises,

(a) mixing a set of primers with a target sample, to produce aprimer-target sample mixture, and incubating the primer-target samplemixture under conditions that promote hybridization between the primersand the target sequence in the primer-target sample mixture, where theprimer-target sample is not subjected to denaturing conditions, and

(b) mixing DNA polymerase with the primer-target sample mixture, toproduce a polymerase-target sample mixture, and incubating thepolymerase-target sample mixture under conditions that promotereplication of the target sequence.

Replication of the target sequence results in replicated strands, whereduring replication at least one of the replicated strands is displacedfrom the target sequence by strand displacement replication of anotherreplicated strand. The target sequence is a nucleic acid sample ofsubstantial complexity, and the set of primers comprises primers havingrandom nucleotide sequences.

Also disclosed is a method of amplifying a target nucleic acid sequence,the method comprising,

(a) mixing a set of primers with a target sample, to produce aprimer-target sample mixture, and incubating the primer-target samplemixture under conditions that promote hybridization between the primersand the target sequence in the primer-target sample mixture, where theprimer-target sample is not subjected to denaturing conditions, and

(b) mixing DNA polymerase with the primer-target sample mixture, toproduce a polymerase-target sample mixture, and incubating thepolymerase-target sample mixture under conditions that promotereplication of the target sequence.

All of the primers in the set of primers are complementary to the samestrand in the target sequence. Replication of the target sequenceresults in replicated strands, where during replication at least one ofthe replicated strands is displaced from the target sequence by stranddisplacement replication of another replicated strand. The set ofprimers can have 3 or more primers.

Also disclosed is a method of amplifying a target nucleic acid sequence,where the method comprises,

(a) mixing a set of primers with a target sample, to produce aprimer-target sample mixture, and incubating the primer-target samplemixture under conditions that promote hybridization between the primersand the target sequence in the primer-target sample mixture, where theprimer-target sample is not subjected to denaturing conditions, and

(b) mixing DNA polymerase with the primer-target sample mixture, toproduce a polymerase-target sample mixture, and incubating thepolymerase-target sample mixture under conditions that promotereplication of the target sequence.

Replication of the target sequence results in replicated strands, whereduring replication at least one of the replicated strands is displacedfrom the target sequence by strand displacement replication of anotherreplicated strand. The target sequence is a nucleic acid sample ofsubstantial complexity, and the set of primers comprises primers havingrandom nucleotide sequences. Each primer comprises a constant portionand a random portion, where the constant portion of each primer has thesame nucleotide sequence and the random portion of each primer has arandom nucleotide sequence.

Also disclosed is a method of amplifying a target nucleic acid sequence,where the method comprises,

(a) mixing a set of primers with a target sample, to produce aprimer-target sample mixture, and incubating the primer-target samplemixture under conditions that promote hybridization between the primersand the target sequence in the primer-target sample mixture, where theprimer-target sample is not subjected to denaturing conditions, and

(b) mixing DNA polymerase with the primer-target sample mixture, toproduce a polymerase-target sample mixture, and incubating thepolymerase-target sample mixture under conditions that promotereplication of the target sequence.

Replication of the target sequence results in replicated strands, whereduring replication at least one of the replicated strands is displacedfrom the target sequence by strand displacement replication of anotherreplicated strand. The conditions that promote replication of the targetsequence do not involve thermal cycling and the target sequence isconcatenated DNA.

Also disclosed is a method of amplifying a target nucleic acid sequence,where the method comprises, bringing into contact a set of primers, DNApolymerase, and a target sample, and incubating the target sample underconditions that promote replication of the target sequence. The targetsample is not subjected to denaturing conditions. Replication of thetarget sequence results in replicated strands, where during replicationat least one of the replicated strands is displaced from the targetsequence by strand displacement replication of another replicatedstrand. The set of primers can have 3 or more primers complementary tothe same strand of the target sequence.

Also disclosed is a method of amplifying a target nucleic acid sequence,where the method comprises, bringing into contact a set of primers, DNApolymerase, and a target sample, and incubating the target sample underconditions that promote replication of the target sequence. The targetsample is not subjected to denaturing conditions. Replication of thetarget sequence results in replicated strands, where during replicationat least one of the replicated strands is displaced from the targetsequence by strand displacement replication of another replicatedstrand. The target sequence is a nucleic acid sample of substantialcomplexity, and the set of primers comprises primers having randomnucleotide sequences.

Also disclosed is a method of amplifying a target nucleic acid sequence,where the method comprises, bringing into contact a set of primers, DNApolymerase, and a target sample, and incubating the target sample underconditions that promote replication of the target sequence. The targetsample is not subjected to denaturing conditions. All of the primers inthe set of primers are complementary to the same strand in the targetsequence. Replication of the target sequence results in replicatedstrands, where during replication at least one of the replicated strandsis displaced from the target sequence by strand displacement replicationof another replicated strand. The set of primers can have 3 or moreprimers.

Also disclosed is a method of amplifying a target nucleic acid sequence,where the method comprises, bringing into contact a set of primers, DNApolymerase, and a target sample, and incubating the target sample underconditions that promote replication of the target sequence. The targetsample is not subjected to denaturing conditions. Replication of thetarget sequence results in replicated strands, where during replicationat least one of the replicated strands is displaced from the targetsequence by strand displacement replication of another replicatedstrand. The target sequence is a nucleic acid sample of substantialcomplexity, and the set of primers comprises primers having randomnucleotide sequences. Each primer comprises a constant portion and arandom portion, where the constant portion of each primer has the samenucleotide sequence and the random portion of each primer has a randomnucleotide sequence.

Also disclosed is a method of amplifying a target nucleic acid sequence,where the method comprises, bringing into contact a set of primers, DNApolymerase, and a target sample, and incubating the target sample underconditions that promote replication of the target sequence. The targetsample is not subjected to denaturing conditions. Replication of thetarget sequence results in replicated strands, where during replicationat least one of the replicated strands is displaced from the targetsequence by strand displacement replication of another replicatedstrand. The conditions that promote replication of the target sequencedo not involve thermal cycling, and the target sequence is concatenatedDNA.

Also disclosed is a method of amplifying a target nucleic acid sequence,where the method comprises, bringing into contact a set of primers, DNApolymerase, and a target sample, and incubating the target sample underconditions that promote replication of the target sequence. The targetsample is not subjected to heat denaturing conditions. Replication ofthe target sequence results in replicated strands, where duringreplication at least one of the replicated strands is displaced from thetarget sequence by strand displacement replication of another replicatedstrand.

Also disclosed is a method of labeling nucleic acids produced by stranddisplacement replication, where the method comprises, labeling nucleicacids produced by strand displacement replication using terminaldeoxynucleotidyl transferase. The replicated strands can be labeled bythe addition of modified nucleotides to the 3′ ends of the nucleicacids. The modified nucleotides can be biotinylated nucleotides,fluorescent nucleotides, 5 methyl dCTP, BrdUTP, or5-(3-aminoallyl)-2′-deoxyuridine 5′-triphosphates.

Also disclosed is a method of labeling nucleic acids produced by stranddisplacement replication, where the method comprises, incorporatingmodified nucleotides into nucleic acids produced by strand displacementreplication during replication.

Also disclosed is a method of attaching nucleic acids produced by stranddisplacement replication, where the method comprises, adding modifiednucleotides to the 3′ ends of nucleic acids produced by stranddisplacement replication using terminal deoxynucleotidyl transferase,and reacting the modified nucleotides with a solid-state support therebyattaching the nucleic acids to the solid-state support.

Also disclosed is a microarray comprising nucleic acids produced bystrand displacement replication coupled or adhered to a solid-statesubstrate.

Also disclosed is a method of generating probes based on a targetnucleic acid sequence, where the method comprises, bringing into contacta set of primers, DNA polymerase, and a target sample, and incubatingthe target sample under conditions that promote replication of thetarget sequence. Replication of the target sequence results inreplicated strands, where during replication at least one of thereplicated strands is displaced from the target sequence by stranddisplacement replication of another replicated strand. The replicatedstrands are used as hybridization probes. The method can furthercomprise labeling the hybridization probes using terminaldeoxynucleotidyl transferase.

Also disclosed is a method of amplifying messenger RNA, where the methodcomprises, reverse transcribing messenger RNA to produce a first strandcDNA, bringing into contact a set of primers, DNA polymerase, and thefirst strand cDNA, and incubating under conditions that promotereplication of the first strand cDNA. Replication of the first strandcDNA results in replicated strands, where during replication at leastone of the replicated strands is displaced from the first strand cDNA bystrand displacement replication of another replicated strand. The methodcan further comprise, prior to replication, degrading the messenger RNAusing RNAse H.

The method can further comprise bringing into contact a set of primers,DNA polymerase, and a second target sample, and incubating the secondtarget sample under conditions that promote replication of the targetsequence. The second target sample is not subjected to denaturingconditions. Replication of the target sequence results in replicatedstrands, where during replication at least one of the replicated strandsis displaced from the target sequence by strand displacement replicationof another replicated strand.

Also disclosed is a method of amplifying a target nucleic acid sequence,where the method comprises, partially degrading RNA in a target sample,bringing into contact DNA polymerase, and the target sample, andincubating the target sample under conditions that promote replicationof the target sequence. Replication of the target sequence results inreplicated strands, where during replication at least one of thereplicated strands is displaced from the target sequence by stranddisplacement replication of another replicated strand.

Also disclosed is a method of comparative genome hybridization, wherethe method comprises, hybridizing nucleic acids produced by stranddisplacement replication of a first sample with nucleic acids producedby strand displacement replication of a second sample. Hybridization canbe carried out in the absence of CotI DNA.

Also disclosed is a method of amplifying a target nucleic acid sequence,where the method comprises, bringing into contact a set of primers, DNApolymerase, and a target sample, and incubating the target sample underconditions that promote replication of the target sequence. A circularnucleic acid molecule comprises the target sequence. Replication of thetarget sequence results in replicated strands, where during replicationat least one of the replicated strands is displaced from the targetsequence by strand displacement replication of another replicatedstrand.

The set of primers can comprise primers having random nucleotidesequences. The primers can be 5, 6, 7, or 8 nucleotides long. Theprimers can be 6, 7, or 8 nucleotides long. The primers can be 6nucleotides long. The modified nucleotides can be biotinylatednucleotides, fluorescent nucleotides, 5 methyl dCTP, BrdUTP, or5-(3-aminoallyl)-2′-deoxyuridine 5′-triphosphates. The modifiednucleotides can be 5-(3-aminoallyl)-2′-deoxyuridine 5′-triphosphates andthe replicated strands can be labeled by reacting labels with theincorporated 5-(3-aminoallyl)-2′-deoxyuridines. The labels can befluorescein isothiocyanate, 5,6-carboxymethyl fluorescein, Texas red,nitrobenz-2-oxa-1,3-diazol-4-yl, coumarin, dansyl chloride, rhodamine,amino-methyl coumarin, Eosin, Erythrosin, BODIPY®, Cascade Blue®, OregonGreen®, pyrene, lissamine, xanthene, acridine, oxazines, phycoerythrin,Cy3, Cy3.5, Cy5, Cy5.5, Cy7, or a combination thereof.

The replicated strands can be used as elements in a microarray. Thereplicated strands can be cleaved prior to use as hybridization probes.The replicated strands can be cleaved with DNAse I. The hybridizationprobes can be labeled by the addition of modified nucleotides to the 3′ends of the replicated strands. The modified nucleotides can be reactedwith a solid-state support thereby attaching the replicated strands tothe solid-state support.

The target sample can be a blood sample, a urine sample, a semen sample,a lymphatic fluid sample, a cerebrospinal fluid sample, amniotic fluidsample, a biopsy sample, a needle aspiration biopsy sample, a cancersample, a tumor sample, a tissue sample, a cell sample, a cell lysatesample, a crude cell lysate sample, a forensic sample, an archeologicalsample, an infection sample, a nosocomial infection sample, a productionsample, a drug preparation sample, a biological molecule productionsample, a protein preparation sample, a lipid preparation sample, acarbohydrate preparation sample, or a combination thereof. The targetsample can be a blood sample. The target sample is a needle aspirationbiopsy sample. The target sample can be a crude cell lysate sample. Thetarget sample can be a nosocomial infection sample. The sample cancontain both human and non-human nucleic acids. The non-human nucleicacid can be amplified preferentially by the use of primers specific forthe non-human nucleic acid. The human nucleic acid can be amplifiedpreferentially by the use of primers specific for human nucleic acid.The sample can be derived from a patient. The target sample can be abiological molecule production sample. Production of replicated strandscan indicate the presence of nucleic acids in the sample. The amount ofreplicated strands produced can indicate the amount of nucleic acids inthe sample. The target sample can be a drug preparation sample. Thetarget sample can be a tumor sample. The target sample can be anamniotic fluid sample. The replicated strands produced from the targetsample can represent a nucleic acid fingerprint of the sample.

The second target sample can be a sample from the same type of organismas the first target sample. The second target sample can be a samplefrom the same type of tissue as the first target sample. The secondtarget sample can be a sample from the same organism as the first targetsample. The second target sample can be obtained at a different timethan the first target sample. The second target sample can be a samplefrom a different organism than the first target sample. The secondtarget sample can be a sample from a different type of tissue than thefirst target sample. The second target sample can be a sample from adifferent species of organism than the first target sample. The secondtarget sample can be a sample from a different strain of organism thanthe first target sample. The second target sample can be a sample from adifferent cellular compartment than the first target sample.

The circular nucleic acid molecule can be produced by digesting genomicDNA with a restriction endonuclease, and circularizing the digested DNA.The digested DNA can be circularized with DNA or RNA ligase. Thedigested DNA can be circularized with a splint or adaptor. The targetsequence can comprises an amplification target and a hybridizationtarget, the hybridization target can flank the amplification target, theset of primers can comprise a plurality of primers, each primer cancomprise a complementary portion, and the complementary portions of theprimers each can be complementary to a different portion of thehybridization target.

The set of primers can comprise a right set of primers and a left set ofprimers, the target sequence can be double-stranded, having a first anda second strand, the hybridization target can comprise a right and lefthybridization target, the right hybridization target can flank theamplification target on one end and the left hybridization target canflank the amplification target on the other end. The complementaryportions of the right set primers can be (i) all complementary to thefirst strand of the target sequence and (ii) each complementary to adifferent portion of the right hybridization target, and thecomplementary portions of the left set primers can be (i) allcomplementary to the second strand of the target sequence and (ii) eachcomplementary to a different portion of the left hybridization target.

The circular nucleic acid molecule can be produced by circularizingcDNA. The circular nucleic acid molecule can be produced bycircularizing mRNA/cDNA hybrid. The mRNA/cDNA hybrid can be circularizedwith DNA or RNA ligase. The mRNA/cDNA hybrid can be circularized with asplint or adaptor.

Also disclosed is a method of amplifying a whole genome, where themethod comprises, bringing into contact a set of primers, DNApolymerase, and a target sample, where the target sample comprises awhole genome, and incubating the target sample under conditions thatpromote replication of the genome. The target sample is not subjected todenaturing conditions. Replication of the genome results in replicatedstrands, where during replication at least one of the replicated strandsis displaced from the genome by strand displacement replication ofanother replicated strand. The primers can be 6 nucleotides long, theprimers each can contain at least one modified nucleotide such that theprimers are nuclease resistant, and the DNA polymerase can be φ29 DNApolymerase. The primers can be of random nucleotide composition, theprimers each can contain at least one modified nucleotide such that theprimers are nuclease resistant, and the DNA polymerase can be φ29 DNApolymerase.

Also disclosed is a method of amplifying a chromosome, where the methodcomprises, bringing into contact a set of primers, DNA polymerase, and atarget sample, where the target sample comprises a chromosome, andincubating the target sample under conditions that promote replicationof the chromosome, where the target sample is not subjected todenaturing conditions. Replication of the chromosome results inreplicated strands, where during replication at least one of thereplicated strands is displaced from the chromosome by stranddisplacement replication of another replicated strand. The primers canbe 6 nucleotides long, the primers each can contain at least onemodified nucleotide such that the primers are nuclease resistant, andthe DNA polymerase can be φ29 DNA polymerase. The primers can be ofrandom nucleotide composition, the primers each can contain at least onemodified nucleotide such that the primers are nuclease resistant, andthe DNA polymerase can be φ29 DNA polymerase.

Disclosed are methods of amplifying a whole genome, the methodscomprising, lysing cells to form a cell lysate, wherein the cell lysatecomprises a whole genome, neutralizing the cell lysate to form aneutralized cell lysate, and incubating the neutralized cell lysateunder conditions that promote replication of the genome, whereinreplication of the genome results in replicated strands, wherein duringreplication at least one of the replicated strands is displaced from thegenome by strand displacement replication of another replicated strand.

Disclosed are methods of amplifying a whole genome, the methodscomprising, lysing cells to form a cell lysate, wherein the cell lysatecomprises a whole genome, neutralizing the cell lysate, wherein the celllysate is not purified, and incubating the cell lysate under conditionsthat promote replication of the genome, wherein replication of thegenome results in replicated strands, wherein during replication atleast one of the replicated strands is displaced from the genome bystrand displacement replication of another replicated strand.

Disclosed are methods of amplifying a whole genome, the methodscomprising, lysing cells to form a cell lysate, wherein the cell lysatecomprises a whole genome, neutralizing the cell lysate, wherein nucleicacids in the cell lysate are not separated from other material in thecell lysate, and incubating the cell lysate under conditions thatpromote replication of the genome, wherein replication of the genomeresults in replicated strands, wherein during replication at least oneof the replicated strands is displaced from the genome by stranddisplacement replication of another replicated strand.

Furthermore, disclosed are methods of amplifying a whole genome, themethods comprising, lysing cells to form a cell lysate, wherein the celllysate comprises a whole genome, neutralizing the cell lysate to form aneutralized cell lysate, and incubating the neutralized cell lysateunder conditions that promote replication of the genome, wherein theneutralized cell lysate is not subjected to denaturing conditions,wherein replication of the genome results in replicated strands, whereinduring replication at least one of the replicated strands is displacedfrom the genome by strand displacement replication of another replicatedstrand.

Also disclosed are methods of amplifying a whole genome, the methodscomprising, lysing cells to form a cell lysate, wherein the cell lysatecomprises a whole genome, neutralizing the cell lysate to form aneutralized cell lysate, and incubating the neutralized cell lysateunder conditions that promote replication of the genome, wherein theneutralized cell lysate is subjected to heat denaturing conditions,wherein replication of the genome results in replicated strands, whereinduring replication at least one of the replicated strands is displacedfrom the genome by strand displacement replication of another replicatedstrand.

Methods of amplifying a whole genome are also disclosed, the methodscomprising, lysing cells to form a cell lysate, wherein the cell lysatecomprises a whole genome, neutralizing the cell lysate to form aneutralized cell lysate, wherein nucleic acids in the neutralized celllysate are not separated from other material in the cell lysate, andincubating the neutralized cell lysate under conditions that promotereplication of the genome, wherein the neutralized cell lysate is notsubjected to denaturing conditions, wherein replication of the genomeresults in replicated strands, wherein during replication at least oneof the replicated strands is displaced from the genome by stranddisplacement replication of another replicated strand.

Disclosed are methods of amplifying a whole genome, the methodscomprising, lysing cells to form a cell lysate, neutralizing the celllysate to form a neutralized cell lysate, and bringing into contact aset of primers, DNA polymerase, and the neutralized cell lysate, whereinthe neutralized cell lysate comprises a whole genome, and incubating theneutralized cell lysate under conditions that promote replication of thegenome, wherein replication of the genome results in replicated strands,wherein during replication at least one of the replicated strands isdisplaced from the genome by strand displacement replication of anotherreplicated strand.

Disclosed are methods of amplifying a whole genome, the methodscomprising, lysing cells to form a cell lysate, neutralizing the celllysate, bringing into contact a set of primers, DNA polymerase, and thecell lysate, wherein the cell lysate comprises a whole genome, whereinthe cell lysate is not purified, and incubating the cell lysate underconditions that promote replication of the genome, wherein replicationof the genome results in replicated strands, wherein during replicationat least one of the replicated strands is displaced from the genome bystrand displacement replication of another replicated strand.

Disclosed are methods of amplifying a whole genome, the methodscomprising, lysing cells to form a cell lysate, neutralizing the celllysate, bringing into contact a set of primers, DNA polymerase, and thecell lysate, wherein the cell lysate comprises a whole genome, whereinnucleic acids in the cell lysate are not separated from other materialin the cell lysate, and incubating the cell lysate under conditions thatpromote replication of the genome, wherein replication of the genomeresults in replicated strands, wherein during replication at least oneof the replicated strands is displaced from the genome by stranddisplacement replication of another replicated strand.

Disclosed are methods of amplifying a whole genome, the methodscomprising, lysing cells to form a cell lysate, neutralizing the celllysate to form a neutralized cell lysate, and bringing into contact aset of primers, DNA polymerase, and the neutralized cell lysate, whereinthe neutralized cell lysate comprises a whole genome, and incubating theneutralized cell lysate under conditions that promote replication of thegenome, wherein the neutralized cell lysate is not subjected todenaturing conditions, wherein replication of the genome results inreplicated strands, wherein during replication at least one of thereplicated strands is displaced from the genome by strand displacementreplication of another replicated strand.

Also disclosed are methods of amplifying a whole genome, the methodscomprising, lysing cells to form a cell lysate, neutralizing the celllysate to form a neutralized cell lysate, and bringing into contact aset of primers, DNA polymerase, and the neutralized cell lysate, whereinthe neutralized cell lysate comprises a whole genome, and incubating theneutralized cell lysate under conditions that promote replication of thegenome, wherein the neutralized cell lysate is subjected to heatdenaturing conditions, wherein replication of the genome results inreplicated strands, wherein during replication at least one of thereplicated strands is displaced from the genome by strand displacementreplication of another replicated strand.

Disclosed are methods of amplifying a whole genome, the methodcomprising, lysing cells to form a cell lysate, neutralizing the celllysate to form a neutralized cell lysate, and bringing into contact aset of primers, DNA polymerase, and the neutralized cell lysate, whereinthe neutralized cell lysate comprises a whole genome, wherein nucleicacids in the neutralized cell lysate are not separated from othermaterial in the neutralized cell lysate, and incubating the neutralizedcell lysate under conditions that promote replication of the genome,wherein the neutralized cell lysate is not subjected to denaturingconditions, wherein replication of the genome results in replicatedstrands, wherein during replication at least one of the replicatedstrands is displaced from the genome by strand displacement replicationof another replicated strand.

Also disclosed are methods of amplifying a target nucleic acid sequence,the method comprising, lysing cells to form a cell lysate, neutralizingthe cell lysate to form a neutralized cell lysate, and incubating theneutralized cell lysate in the presence of a set of primers and DNApolymerase and under conditions that promote replication of a targetsequence, wherein replication of the neutralized cell lysate results inreplicated strands, wherein during replication at least one of thereplicated strands is displaced from the target sequence by stranddisplacement replication of another replicated strand.

Disclosed are methods of amplifying a target nucleic acid sequence, themethods comprising, lysing cells to form a cell lysate, neutralizing thecell lysate, and incubating the cell lysate in the presence of a set ofprimers and DNA polymerase and under conditions that promote replicationof a target sequence, wherein the cell lysate is not purified, whereinreplication of the cell lysate results in replicated strands, whereinduring replication at least one of the replicated strands is displacedfrom the target sequence by strand displacement replication of anotherreplicated strand.

Disclosed are methods of amplifying a target nucleic acid sequence, themethods comprising, lysing cells to form a cell lysate, neutralizing thecell lysate, and incubating the cell lysate in the presence of a set ofprimers and DNA polymerase and under conditions that promote replicationof a target sequence, wherein nucleic acids in the cell lysate are notseparated from other material in the cell lysate, wherein replication ofthe cell lysate results in replicated strands, wherein duringreplication at least one of the replicated strands is displaced from thetarget sequence by strand displacement replication of another replicatedstrand.

Also disclosed are methods of amplifying a target nucleic acid sequence,the methods comprising, lysing cells to form a cell lysate, neutralizingthe cell lysate to form a neutralized cell lysate, and incubating theneutralized cell lysate in the presence of a set of primers and DNApolymerase and under conditions that promote replication of a targetsequence, wherein the neutralized cell lysate is not subjected todenaturing conditions, wherein replication of the neutralized celllysate results in replicated strands, wherein during replication atleast one of the replicated strands is displaced from the targetsequence by strand displacement replication of another replicatedstrand.

Disclosed are methods of amplifying a target nucleic acid sequence, themethods comprising, lysing cells to form a cell lysate, neutralizing thecell lysate to form a neutralized cell lysate, and incubating theneutralized cell lysate in the presence of a set of primers and DNApolymerase and under conditions that promote replication of a targetsequence, wherein the neutralized cell lysate is subjected to denaturingconditions, wherein replication of the neutralized cell lysate resultsin replicated strands, wherein during replication at least one of thereplicated strands is displaced from the target sequence by stranddisplacement replication of another replicated strand.

Disclosed are methods of amplifying a target nucleic acid sequence, themethods comprising, lysing cells to form a cell lysate, neutralizing thecell lysate to form a neutralized cell lysate, and incubating theneutralized cell lysate in the presence of a set of primers and DNApolymerase and under conditions that promote replication of a targetsequence, wherein nucleic acids in the neutralized cell lysate are notseparated from other material in the neutralized cell lysate, whereinthe neutralized cell lysate is not subjected to denaturing conditions,wherein replication of the neutralized cell lysate results in replicatedstrands, wherein during replication at least one of the replicatedstrands is displaced from the target sequence by strand displacementreplication of another replicated strand.

Also disclosed are methods of amplifying a target nucleic acid sequence,the methods comprising, lysing cells to form a cell lysate, neutralizingthe cell lysate to form a neutralized cell lysate, and bringing intocontact a set of primers, DNA polymerase, and the neutralized celllysate, and incubating the neutralized cell lysate under conditions thatpromote replication of a target sequence, wherein replication of theneutralized cell lysate results in replicated strands, wherein duringreplication at least one of the replicated strands is displaced from thetarget sequence by strand displacement replication of another replicatedstrand.

Disclosed are methods of amplifying a target nucleic acid sequence, themethods comprising, lysing cells to form a cell lysate, neutralizing thecell lysate, bringing into contact a set of primers, DNA polymerase, andthe cell lysate, and incubating the cell lysate under conditions thatpromote replication of a target sequence, wherein the cell lysate is notpurified, wherein replication of the cell lysate results in replicatedstrands, wherein during replication at least one of the replicatedstrands is displaced from the target sequence by strand displacementreplication of another replicated strand.

Also disclosed are methods of amplifying a target nucleic acid sequence,the methods comprising, lysing cells to form a cell lysate, neutralizingthe cell lysate, bringing into contact a set of primers, DNA polymerase,and the cell lysate, and incubating the cell lysate under conditionsthat promote replication of a target sequence, wherein nucleic acids inthe cell lysate are not separated from other material in the celllysate, wherein replication of the cell lysate results in replicatedstrands, wherein during replication at least one of the replicatedstrands is displaced from the target sequence by strand displacementreplication of another replicated strand.

Disclosed are methods of amplifying a target nucleic acid sequence, themethods comprising, lysing cells to form a cell lysate, neutralizing thecell lysate to form a neutralized cell lysate, and bringing into contacta set of primers, DNA polymerase, and the neutralized cell lysate, andincubating the neutralized cell lysate under conditions that promotereplication of a target sequence, wherein the neutralized cell lysate isnot subjected to denaturing conditions, wherein replication of theneutralized cell lysate results in replicated strands, wherein duringreplication at least one of the replicated strands is displaced from thetarget sequence by strand displacement replication of another replicatedstrand.

Disclosed are methods of amplifying a target nucleic acid sequence, themethods comprising, lysing cells to form a cell lysate, neutralizing thecell lysate to form a neutralized cell lysate, and bringing into contacta set of primers, DNA polymerase, and the neutralized cell lysate, andincubating the neutralized cell lysate under conditions that promotereplication of a target sequence, wherein the neutralized cell lysate issubjected to heat denaturing conditions, wherein replication of theneutralized cell lysate results in replicated strands, wherein duringreplication at least one of the replicated strands is displaced from thetarget sequence by strand displacement replication of another replicatedstrand.

Disclosed are methods of amplifying a target nucleic acid sequence, themethod comprising, lysing cells to form a cell lysate, neutralizing thecell lysate to form a neutralized cell lysate, and bringing into contacta set of primers, DNA polymerase, and the neutralized cell lysate, andincubating the neutralized cell lysate under conditions that promotereplication of a target sequence, wherein nucleic acids in theneutralized cell lysate are not separated from other material in theneutralized cell lysate, wherein the neutralized cell lysate is notsubjected to denaturing conditions, wherein replication of theneutralized cell lysate results in replicated strands, wherein duringreplication at least one of the replicated strands is displaced from thetarget sequence by strand displacement replication of another replicatedstrand.

Disclosed are methods of amplifying a whole genome, the methodcomprising, lysing cells to form a cell lysate, wherein the cell lysatecomprises a whole genome, and incubating the cell lysate underconditions that promote replication of the genome, wherein replicationof the genome results in replicated strands, wherein during replicationat least one of the replicated strands is displaced from the genome bystrand displacement replication of another replicated strand.

Also disclosed are methods of amplifying a whole genome, the methodscomprising, lysing cells to form a cell lysate, wherein the cell lysatecomprises a whole genome, wherein the cell lysate is not purified, andincubating the cell lysate under conditions that promote replication ofthe genome, wherein replication of the genome results in replicatedstrands, wherein during replication at least one of the replicatedstrands is displaced from the genome by strand displacement replicationof another replicated strand.

Disclosed are methods of amplifying a whole genome, the methodscomprising, lysing cells to form a cell lysate, wherein the cell lysatecomprises a whole genome, wherein nucleic acids in the cell lysate arenot separated from other material in the cell lysate, and incubating thecell lysate under conditions that promote replication of the genome,wherein replication of the genome results in replicated strands, whereinduring replication at least one of the replicated strands is displacedfrom the genome by strand displacement replication of another replicatedstrand.

Disclosed are methods of amplifying a whole genome, the methodscomprising, lysing cells to form a cell lysate, wherein the cell lysatecomprises a whole genome, and incubating the cell lysate underconditions that promote replication of the genome, wherein the celllysate is not subjected to denaturing conditions, wherein replication ofthe genome results in replicated strands, wherein during replication atleast one of the replicated strands is displaced from the genome bystrand displacement replication of another replicated strand.

Also disclosed are methods of amplifying a whole genome, the methodscomprising, lysing cells to form a cell lysate, wherein the cell lysatecomprises a whole genome, and incubating the cell lysate underconditions that promote replication of the genome, wherein the celllysate is subjected to heat denaturing conditions, wherein replicationof the genome results in replicated strands, wherein during replicationat least one of the replicated strands is displaced from the genome bystrand displacement replication of another replicated strand.

Disclosed are methods of amplifying a whole genome, the methodcomprising, lysing cells to form a cell lysate, wherein the cell lysatecomprises a whole genome, wherein nucleic acids in the cell lysate arenot separated from other material in the cell lysate, and incubating thecell lysate under conditions that promote replication of the genome,wherein the cell lysate is not subjected to denaturing conditions,wherein replication of the genome results in replicated strands, whereinduring replication at least one of the replicated strands is displacedfrom the genome by strand displacement replication of another replicatedstrand.

Also disclosed are methods of amplifying a whole genome, the methodcomprising, lysing cells to form a cell lysate, bringing into contact aset of primers, DNA polymerase, and the cell lysate, wherein the celllysate comprises a whole genome, and incubating the cell lysate underconditions that promote replication of the genome, wherein replicationof the genome results in replicated strands, wherein during replicationat least one of the replicated strands is displaced from the genome bystrand displacement replication of another replicated strand.

Disclosed are methods of amplifying a whole genome, the methodscomprising, lysing cells to form a cell lysate, bringing into contact aset of primers, DNA polymerase, and the cell lysate, wherein the celllysate comprises a whole genome, wherein the cell lysate is notpurified, and incubating the cell lysate under conditions that promotereplication of the genome, wherein replication of the genome results inreplicated strands, wherein during replication at least one of thereplicated strands is displaced from the genome by strand displacementreplication of another replicated strand.

Disclosed are methods of amplifying a whole genome, the methodscomprising, lysing cells to form a cell lysate, bringing into contact aset of primers, DNA polymerase, and the cell lysate, wherein the celllysate comprises a whole genome, wherein nucleic acids in the celllysate are not separated from other material in the cell lysate, andincubating the cell lysate under conditions that promote replication ofthe genome, wherein replication of the genome results in replicatedstrands, wherein during replication at least one of the replicatedstrands is displaced from the genome by strand displacement replicationof another replicated strand.

Also disclosed are methods of amplifying a whole genome, the methodscomprising, lysing cells to form a cell lysate, bringing into contact aset of primers, DNA polymerase, and the cell lysate, wherein the celllysate comprises a whole genome, and incubating the cell lysate underconditions that promote replication of the genome, wherein the celllysate is not subjected to denaturing conditions, wherein replication ofthe genome results in replicated strands, wherein during replication atleast one of the replicated strands is displaced from the genome bystrand displacement replication of another replicated strand.

Disclosed are methods of amplifying a whole genome, the methodscomprising, lysing cells to form a cell lysate, bringing into contact aset of primers, DNA polymerase, and the cell lysate, wherein the celllysate comprises a whole genome, and incubating the cell lysate underconditions that promote replication of the genome, wherein the celllysate is subjected to heat denaturing conditions, wherein replicationof the genome results in replicated strands, wherein during replicationat least one of the replicated strands is displaced from the genome bystrand displacement replication of another replicated strand.

Disclosed are methods of amplifying a whole genome, the methodscomprising, lysing cells to form a cell lysate, bringing into contact aset of primers, DNA polymerase, and the cell lysate, wherein the celllysate comprises a whole genome, wherein nucleic acids in the celllysate are not separated from other material in the cell lysate, andincubating the cell lysate under conditions that promote replication ofthe genome, wherein the cell lysate is not subjected to denaturingconditions, wherein replication of the genome results in replicatedstrands, wherein during replication at least one of the replicatedstrands is displaced from the genome by strand displacement replicationof another replicated strand.

Also disclosed are methods of amplifying a target nucleic acid sequence,the methods comprising, lysing cells to form a cell lysate, andincubating the cell lysate in the presence of a set of primers and DNApolymerase and under conditions that promote replication of a targetsequence, wherein replication of the cell lysate results in replicatedstrands, wherein during replication at least one of the replicatedstrands is displaced from the target sequence by strand displacementreplication of another replicated strand.

Disclosed are methods of amplifying a target nucleic acid sequence, themethods comprising, lysing cells to form a cell lysate, and incubatingthe cell lysate in the presence of a set of primers and DNA polymeraseand under conditions that promote replication of a target sequence,wherein the cell lysate is not purified, wherein replication of the celllysate results in replicated strands, wherein during replication atleast one of the replicated strands is displaced from the targetsequence by strand displacement replication of another replicatedstrand.

Also disclosed are methods of amplifying a target nucleic acid sequence,the methods comprising, lysing cells to form a cell lysate, andincubating the cell lysate in the presence of a set of primers and DNApolymerase and under conditions that promote replication of a targetsequence, wherein nucleic acids in the cell lysate are not separatedfrom other material in the cell lysate, wherein replication of the celllysate results in replicated strands, wherein during replication atleast one of the replicated strands is displaced from the targetsequence by strand displacement replication of another replicatedstrand.

Disclosed are methods of amplifying a target nucleic acid sequence, themethods comprising, lysing cells to form a cell lysate, and incubatingthe cell lysate in the presence of a set of primers and DNA polymeraseand under conditions that promote replication of a target sequence,wherein the cell lysate is not subjected to denaturing conditions,wherein replication of the cell lysate results in replicated strands,wherein during replication at least one of the replicated strands isdisplaced from the target sequence by strand displacement replication ofanother replicated strand.

Also disclosed are methods of amplifying a target nucleic acid sequence,the method comprising, lysing cells to form a cell lysate, andincubating the cell lysate in the presence of a set of primers and DNApolymerase and under conditions that promote replication of a targetsequence, wherein the cell lysate is subjected to heat denaturingconditions, wherein replication of the cell lysate results in replicatedstrands, wherein during replication at least one of the replicatedstrands is displaced from the target sequence by strand displacementreplication of another replicated strand.

Disclosed are methods of amplifying a target nucleic acid sequence, themethod comprising, lysing cells to form a cell lysate, and incubatingthe cell lysate in the presence of a set of primers and DNA polymeraseand under conditions that promote replication of a target sequence,wherein nucleic acids in the cell lysate are not separated from othermaterial in the cell lysate, wherein the cell lysate is not subjected todenaturing conditions, wherein replication of the cell lysate results inreplicated strands, wherein during replication at least one of thereplicated strands is displaced from the target sequence by stranddisplacement replication of another replicated strand.

Disclosed are methods of amplifying a target nucleic acid sequence, themethod comprising, lysing cells to form a cell lysate, bringing intocontact a set of primers, DNA polymerase, and the cell lysate, andincubating the cell lysate under conditions that promote replication ofa target sequence, wherein replication of the cell lysate results inreplicated strands, wherein during replication at least one of thereplicated strands is displaced from the target sequence by stranddisplacement replication of another replicated strand.

Disclosed are methods of amplifying a target nucleic acid sequence, themethods comprising, lysing cells to form a cell lysate, bringing intocontact a set of primers, DNA polymerase, and the cell lysate, andincubating the cell lysate under conditions that promote replication ofa target sequence, wherein the cell lysate is not purified, whereinreplication of the cell lysate results in replicated strands, whereinduring replication at least one of the replicated strands is displacedfrom the target sequence by strand displacement replication of anotherreplicated strand.

Disclosed are methods of amplifying a target nucleic acid sequence, themethods comprising, lysing cells to form a cell lysate, bringing intocontact a set of primers, DNA polymerase, and the cell lysate, andincubating the cell lysate under conditions that promote replication ofa target sequence, wherein nucleic acids in the cell lysate are notseparated from other material in the cell lysate, wherein replication ofthe cell lysate results in replicated strands, wherein duringreplication at least one of the replicated strands is displaced from thetarget sequence by strand displacement replication of another replicatedstrand.

Also disclosed are methods of amplifying a target nucleic acid sequence,the methods comprising, lysing cells to form a cell lysate, bringinginto contact a set of primers, DNA polymerase, and the cell lysate, andincubating the cell lysate under conditions that promote replication ofa target sequence, wherein the cell lysate is not subjected todenaturing conditions, wherein replication of the cell lysate results inreplicated strands, wherein during replication at least one of thereplicated strands is displaced from the target sequence by stranddisplacement replication of another replicated strand.

Disclosed are methods of amplifying a target nucleic acid sequence, themethods comprising, lysing cells to form a cell lysate, bringing intocontact a set of primers, DNA polymerase, and the cell lysate, andincubating the cell lysate under conditions that promote replication ofa target sequence, wherein the cell lysate is subjected to heatdenaturing conditions, wherein replication of the cell lysate results inreplicated strands, wherein during replication at least one of thereplicated strands is displaced from the target sequence by stranddisplacement replication of another replicated strand.

Disclosed are methods of amplifying a target nucleic acid sequence, themethods comprising, lysing cells to form a cell lysate, bringing intocontact a set of primers, DNA polymerase, and the cell lysate, andincubating the cell lysate under conditions that promote replication ofa target sequence, wherein nucleic acids in the cell lysate are notseparated from other material in the cell lysate, wherein the celllysate is not subjected to denaturing conditions, wherein replication ofthe cell lysate results in replicated strands, wherein duringreplication at least one of the replicated strands is displaced from thetarget sequence by strand displacement replication of another replicatedstrand.

Any of the disclosed methods can be performed wherein the neutralizedcell lysate is not subjected to denaturing conditions or wherein theneutralized cell lysate is subjected to heat denaturing conditions.

Disclosed are kits for amplifying a whole genome, the kits comprising asolution for lysis, a solution for neutralization, a set of primers, anda DNA polymerase.

Also disclosed are kits for amplifying a whole genome, the kitscomprising a solution for cell lysis, a solution for neutralization of acell lysate, a set of primers, and a DNA polymerase.

Disclosed are kits for amplifying a whole genome, the kits comprising asolution for lysing cells, a solution for neutralizing lysed cells, aset of primers, and a DNA polymerase.

Also disclosed are kits for amplifying a whole genome, the kitscomprising a composition for lysis, a composition for neutralization, aset of primers, and a DNA polymerase.

Disclosed are kits for amplifying a whole genome, the kits comprising acomposition for cell lysis, a composition for neutralization of a celllysate, a set of primers, and a DNA polymerase.

Disclosed are kits for amplifying a whole genome, the kits comprising acomposition for lysing cells, a composition for neutralizing lysedcells, a set of primers, and a DNA polymerase.

Disclosed are kits for amplifying a whole genome, the kits comprising asolution for lysis, wherein the solution for lysis is alkaline, asolution for neutralization, a set of primers, wherein the primers are 6nucleotides long, wherein the primers each contain at least one modifiednucleotide such that the primers are nuclease resistant, and a DNApolymerase, wherein the DNA polymerase is φ29 DNA polymerase.

Disclosed are kits for amplifying a whole genome, the kits comprising asolution for cell lysis, wherein the solution for cell lysis isalkaline, a solution for neutralization of a cell lysate, a set ofprimers, wherein the primers are 6 nucleotides long, wherein the primerseach contain at least one modified nucleotide such that the primers arenuclease resistant, and a DNA polymerase, wherein the DNA polymerase isφ29 DNA polymerase.

Also disclosed are kits for amplifying a whole genome, the kitscomprising a solution for lysing cells, wherein the solution for lysingcells is alkaline, a solution for neutralizing lysed cells, a set ofprimers, wherein the primers are 6 nucleotides long, wherein the primerseach contain at least one modified nucleotide such that the primers arenuclease resistant, and a DNA polymerase, wherein the DNA polymerase isφ29 DNA polymerase.

Disclosed are methods of amplifying a whole genome, the methodcomprising, exposing cells to alkaline conditions to form a cell lysate,wherein the cell lysate comprises a whole genome, reducing the pH of thecell lysate to form a stabilized cell lysate, and incubating thestabilized cell lysate under conditions that promote replication of thegenome, wherein replication of the genome results in replicated strands,wherein during replication at least one of the replicated strands isdisplaced from the genome by strand displacement replication of anotherreplicated strand.

Also disclosed are methods, wherein the cells are exposed to alkalineconditions by mixing the cells with a lysis solution or wherein thelysis solution comprises a base or wherein the base is an aqueous base.

Also disclosed are methods, wherein the base is potassium hydroxide,sodium hydroxide, potassium acetate, sodium acetate, ammonium hydroxide,lithium hydroxide, calcium hydroxide, magnesium hydroxide, sodiumcarbonate, sodium bicarbonate, calcium carbonate, ammonia, aniline,benzylamine, n-butylamine, diethylamine, dimethylamine, diphenylamine,ethylamine, ethylenediamine, methylamine, N-methylaniline, morpholine,pyridine, triethylamine, trimethylamine, aluminum hydroxide, rubidiumhydroxide, cesium hydroxide, strontium hydroxide, barium hydroxide, orDBU (1,8-diazobicyclo[5,4,0]undec-7-ene).

Disclosed are methods, wherein the base is potassium hydroxide.

Also disclosed are methods, wherein the lysis solution comprises 400 mMKOH and/or wherein the lysis solution comprises 100 mM dithiothreitol,and 10 mM EDTA or wherein the lysis solution consists of 400 mM KOH, 100mM dithiothreitol, and 10 mM EDTA.

Disclosed are methods, wherein the lysis solution comprises a pluralityof basic agents and/or wherein the cells are mixed with an equal volumeof the lysis solution.

Disclosed are methods, wherein the lysis solution comprises a buffer, aswell as methods wherein the buffer is a phosphate buffer, Good buffer,BES, BICINE, CAPS, EPPS, HEPES, MES, MOPS, PIPES, TAPS, TES, TRICINE,sodium cacodylate, sodium citrate, triethylammonium acetate,triethylammonium bicarbonate, Tris, Bis-tris, or Bis-tris propane. Alsodisclosed are methods, wherein the buffer is Tris-HCl at pH 4.1. Thedisclosed methods can comprises a plurality of buffering agents.

Also disclosed are methods, wherein the pH of the cell lysate is reducedto the range of about pH 7.0 to about pH 6.8.

Also disclosed are methods, wherein the pH of the cell lysate is reducedby mixing the cell lysate with a stabilization solution. Disclosed aremethods wherein the stabilization solution comprises 800 mM Tris-HCl, pH4.1 as well as methods, wherein the stabilization solution comprises aplurality of buffering agents and/or wherein the cell lysate is mixedwith an equal volume of the stabilization solution.

Disclosed are methods, wherein the stabilization solution comprises anacid as well as methods wherein the acid is hydrochloric acid, sulfuricacid, phosphoric acid, acetic acid, acetylsalicylic acid, ascorbic acid,carbonic acid, citric acid, formic acid, nitric acid, perchloric acid,HF, HBr, HI, H₂S, HCN, HSCN, HClO, monochloroacetic acid, dichloroaceticacid, trichloroacetic acid, or a carboxylic acid. Disclosed are methodswherein the carboxylic acid is ethanoic, propanoic, or butanoic acid.Disclosed are methods, wherein the stabilization solution comprises aplurality of acidic agents.

Disclosed are methods, wherein the pH of the cell lysate is reduced toabout pH 9.0 or below, about pH 8.5 or below about pH 8.0 or below, orabout pH 7.5 or below.

Also disclosed are methods, wherein the pH of the cell lysate is reducedto the range of about pH 9.0 to about pH 6.0, about pH 9.0 to about pH7.0, about pH 9.0 to about pH 7.5, pH 9.0 to about pH 8.0, pH 8.5 toabout pH 6.0, pH 8.5 to about pH 7.0, pH 8.5 to about pH 7.5, pH 8.5 toabout pH 8.0, pH 8.0 to about pH 6.0, pH 8.0 to about pH 6.5, pH 8.0 toabout pH 7.0, pH 8.0 to about pH 7.5, pH 7.5 to about pH 6.0, or pH 7.5to about pH 7.0.

Disclosed are methods, wherein nucleic acids in the cell lysate and thestabilized cell lysate are not separated from other material in the celllysate, wherein the cell lysate and the stabilized cell lysate are notsubjected to purification prior to the incubation, wherein thepurification comprises separation of nucleic acids in the cell lysatefrom other material in the cell lysate, wherein the purificationcomprises centrifugation, extraction, chromatography, filtration,dialysis, or a combination of these, wherein the purification comprisesprecipitation other than precipitation caused by the alkaline conditionsor by the reduction of the pH, wherein the purification comprisescentrifugation, phenol-chloroform extraction, column chromatography, ora combination of these, wherein the cell lysate, stabilized cell lysate,or both are subjected to partial purification prior to the incubation,wherein the partial purification comprises centrifugation, extraction,chromatography, precipitation, filtration, dialysis, or a combination ofthese, wherein the partial purification comprises centrifugation,phenol-chloroform extraction, column chromatography, or a combination ofthese, wherein the cell lysate and the stabilized cell lysate are notsubjected to substantial purification prior to the incubation, whereinthe substantial purification does not include centrifugation,extraction, chromatography, precipitation, filtration, or dialysis,wherein the substantial purification does not include centrifugation,phenol-chloroform extraction, or column chromatography, wherein the celllysate, stabilized cell lysate, or both are subjected to centrifugation,extraction, chromatography, precipitation, filtration, or dialysis priorto the incubation, wherein the cell lysate, stabilized cell lysate, orboth are subjected to centrifugation, phenol-chloroform extraction, orcolumn chromatography prior to the incubation, wherein the substantialpurification comprises centrifugation, extraction, chromatography,filtration, dialysis, or a combination of these, wherein the substantialpurification comprises precipitation other than precipitation caused bythe alkaline conditions or by the reduction of the pH, wherein thesubstantial purification comprises centrifugation, phenol-chloroformextraction, column chromatography, or a combination of these, whereinthe cell lysate and the stabilized cell lysate are not purified prior tothe incubation, wherein the cell lysate, stabilized cell lysate, or bothare partially purified prior to the incubation, wherein the incubationis substantially isothermic, wherein neither the cell lysate nor thestabilized cell lysate is heated substantially above the temperature ofthe incubation, wherein neither the cell lysate nor the stabilized celllysate is subjected to substantial heating above the temperature of theincubation, wherein the cells are not heated substantially above thetemperature of the incubation, wherein the cells are not subjected tosubstantial heating above the temperature of the incubation, wherein thecells are not heated substantially above the temperature at which thecells grow, wherein the cells are not subjected to substantial heatingabove the temperature at which the cells grow, wherein the cell lysate,stabilized cell lysate, and the cells are not heated substantially abovethe temperature of the incubation, wherein the cell lysate, stabilizedcell lysate, and the cells are not subjected to substantial heatingabove the temperature of the incubation, wherein the cell lysate,stabilized cell lysate, and the cells are not heated, prior to or duringthe incubation, substantially above the temperature at which the cellsgrow, wherein the cell lysate, stabilized cell lysate, and the cells arenot subjected to, prior to or during the incubation, substantial heatingabove the temperature at which the cells grow prior, wherein neither thecell lysate nor the stabilized cell lysate is heated above a temperatureand for a time that would cause notable denaturation of the genome,wherein neither the cell lysate nor the stabilized cell lysate issubjected to heating above a temperature and for a time that would causenotable denaturation of the genome, wherein the cells are not lysed byheat, wherein the cells are not heated above a temperature and for atime that would cause substantial cell lysis in the absence of thealkaline conditions, and/or wherein the cells are not subjected toheating above a temperature and for a time that would cause substantialcell lysis in the absence of the alkaline conditions.

Disclosed are methods of amplifying a whole genome, the methodscomprising, exposing cells to alkaline conditions to form a cell lysate,wherein the cell lysate comprises a whole genome, wherein the cells areexposed to alkaline conditions by mixing the cells with a lysissolution, reducing the pH of the cell lysate to form a stabilized celllysate, wherein the pH of the cell lysate is reduced by mixing the celllysate with a stabilization solution, and incubating the stabilized celllysate under conditions that promote replication of the genome, whereinreplication of the genome results in replicated strands, wherein duringreplication at least one of the replicated strands is displaced from thegenome by strand displacement replication of another replicated strand

Disclosed are methods, wherein the lysis solution comprises potassiumhydroxide, such as 400 mM KOH.

Also disclosed are methods wherein the lysis solution comprises 400 mMKOH, 100 mM dithiothreitol, and 10 mM EDTA or wherein the lysis solutionconsists of 400 mM KOH, 100 mM dithiothreitol, and 10 mM EDTA.

Disclosed are methods, wherein the cells are mixed with an equal volumeof the lysis solution and/or an equal volume of the stabilizationsolution

Disclosed are methods, wherein the stabilization solution comprisesTris-HCl at pH 4.1 and wherein the stabilization solution comprises 800mM Tris-HCl, pH 4.1.

Also disclosed are methods wherein the stabilization solution consistsof 800 mM Tris-HCl, pH 4.1.

Disclosed are methods, wherein the lysis solution consists of 400 mM KOHand 10 mM EDTA, wherein the stabilization solution consists of 800 mMTris-HCl, pH 4, wherein the stabilized cell lysate is incubated in thepresence of 37.5 mM Tris-HCl, 50 mM KCl, 10 mM MgCl₂, 5 mM (NH₄)₂SO₄, 1mM deoxynucleotide triphosphates, 50 μM primers, and φ29 DNA Polymerase.

Also disclosed are methods, wherein the stabilized cell lysate isincubated in the presence of 37.5 mM Tris-HCl, 50 mM KCl, 10 mM MgCl₂, 5mM (NH₄)₂SO₄, 1 mM deoxynucleotide triphosphates, 50 μM primers, and φ29DNA Polymerase by mixing the stabilized cell lysate with one quartervolume of reaction mix, and φ29 DNA Polymerase, wherein the reaction mixconsists of 150 mM Tris-HCl, 200 mM KCl, 40 mM MgCl₂, 20 mM (NH₄)₂SO₄, 4mM deoxynucleotide triphosphates, and 0.2 mM primers.

Disclosed are methods of amplifying a whole genome, the methodscomprising, exposing cells to alkaline conditions to from a cell lysate,wherein the cell lysate comprises a whole genome, wherein the cells areexposed to alkaline conditions by mixing the cells with a lysissolution, wherein the lysis solution comprises 400 mM KOH, 100 mMdithiothreitol, and 10 mM EDTA, reducing the pH of the cell lysate toform a stabilized cell lysate, wherein the pH of the cell lysate isreduced by mixing the cell lysate with a stabilization solution, whereinthe stabilization solution comprises 800 mM Tris-HCl, pH 4.1, andincubating the stabilized cell lysate under conditions that promotereplication of the genome, wherein replication of the genome results inreplicated strands, wherein during replication at least one of thereplicated strands is displaced from the genome by strand displacementreplication of another replicated strand.

Disclosed are methods, wherein the cells are mixed with an equal volumeof the lysis solution and/or wherein the cell lysate is mixed with anequal volume of the stabilization solution.

Disclosed are kits for amplifying a whole genome, the kits comprising alysis solution, a stabilization solution, a set of primers, and a DNApolymerase.

Disclosed are kits wherein the lysis solution comprises potassiumhydroxide, such as 400 mM KOH.

Also disclosed are kits wherein the lysis solution comprises 400 mM KOH,100 mM dithiothreitol, and-10 mM EDTA.

Disclosed are kits, wherein the stabilization solution comprisesTris-HCl at pH 4.1, wherein the stabilization solution comprises 800 mMTris-HCl, pH 4.1, or wherein the stabilization solution consists of 800mM Tris-HCl, pH 4.1.

Disclosed are kits comprising deoxynucleotide triphosphates.

Disclosed are kits comprising IM dithiotheitol, 1× Phosphase-BufferedSaline, pH 7.5, and control DNA template, wherein the lysis solutioncomprises 400 mM KOH and 10 mM EDTA, wherein the stabilization solutioncomprises 800 mM Tris-HCl, pH 4, wherein the set of primers comprises areaction mix, wherein the reaction mix comprises 150 mM Tris-HCl, 200 mMKCl, 40 mM MgCl₂, 20 mM (NH₄)₂SO₄, 4 mM deoxynucleotide triphosphates,and 0.2 mM random hexamer primers, wherein the DNA polymerase is φ29 DNApolymerase.

Disclosed are kits comprising one or more detection probes, wherein thedetection probes each comprise a complementary portion, wherein thecomplementary portion is complementary to a nucleic acid sequence ofinterest.

Disclosed are kits, wherein the kit is designed to detect nucleic acidsequences of interest in the genome and/or kits wherein the kit isdesigned to assess a disease, condition or predisposition of anindividual based on the nucleic acid sequences of interest.

Disclosed are methods of amplifying damaged DNA, the method comprisingexposing a damaged DNA sample to conditions that promote substantialdenaturation of damaged DNA in the damaged DNA sample, thereby forming adenatured damaged DNA sample, altering the conditions to conditions thatdo not promote substantial denaturation of damaged DNA in the damagedDNA sample to form a stabilized damaged DNA sample, incubating damagedDNA in the stabilized damaged DNA sample under conditions that promotereplication of the damaged DNA, wherein replication of the damaged DNAresults in a longer average fragment length for the replicated damagedDNA than the average fragment length in the damaged DNA sample, whereinduring replication at least one of the replicated strands is displacedby strand displacement replication of another replicated strand.

Disclosed are methods, wherein the damaged DNA sample, the denatureddamaged DNA sample, or both are exposed to ionic conditions, wherein thedamaged DNA sample and denatured damaged DNA sample are exposed to ionicconditions by mixing an ionic solution with the damaged DNA sample,wherein the ionic solution is mixed with the damaged DNA sample prior toor during exposure of the damaged DNA sample to conditions that promotesubstantial denaturation of the damaged DNA, wherein the ionic solutionis a salt solution, wherein the salt solution comprises one or moresalts, wherein the salt is Tris-HCl, Tris-EDTA, sodium chloride,potassium chloride, magnesium chloride, sodium acetate, potassiumacetate, magnesium acetate, or a combination, wherein the Tris-HCl isfrom pH 7.0 to 8.0, wherein the salt is Tris-EDTA, wherein the saltsolution comprises about 50 mM to about 500 mM Tris and about 1 mM toabout 5 mM EDTA, wherein the ionic solution is diluted 2 to 5 fold whenmixed with the damaged DNA sample, wherein the denatured damaged DNAsample is exposed to ionic conditions by mixing an ionic solution withthe denatured damaged DNA sample, wherein the ionic solution is mixedwith the denatured damaged DNA sample prior to or during altering of theconditions, wherein the damaged DNA sample is exposed to conditions thatpromote substantial denaturation by mixing the damaged DNA sample with adenaturing solution and by heating the damaged DNA sample to atemperature and for a length of time that substantially denatures thedamaged DNA in the damaged DNA sample, wherein the damaged DNA sample ismixed with the denaturing solution after the DNA sample is heated,wherein the damaged DNA sample is mixed with the denaturing solutionbefore the DNA sample is heated, wherein the damaged DNA sample is mixedwith the denaturing solution at the same time the DNA sample is heated,wherein the damaged DNA sample is mixed with the denaturing solutionduring heating of the DNA sample, wherein the damaged DNA sample ismixed with the denaturing solution when heating of the DNA samplebegins, and/or wherein mixing the damaged DNA sample with a denaturingsolution produces alkaline conditions in the damaged DNA sample

Disclosed are methods wherein the denaturing solution comprises a base,wherein the base is an aqueous base, wherein the base is sodiumhydroxide, potassium hydroxide, potassium acetate, sodium acetate,ammonium hydroxide, lithium hydroxide, calcium hydroxide, magnesiumhydroxide, sodium carbonate, sodium bicarbonate, calcium carbonate,ammonia, aniline, benzylamine, n-butylamine, diethylamine,dimethylamine, diphenylamine, ethylamine, ethylenediamine, methylamine,N-methylaniline, morpholine, pyridine, triethylamine, trimethylamine,aluminum hydroxide, rubidium hydroxide, cesium hydroxide, strontiumhydroxide, barium hydroxide, or DBU(1,8-diazobicyclo[5,4,0]undec-7-ene), wherein the base is sodiumhydroxide, wherein the denaturing solution comprises about 150 mM toabout 1 M NaOH.

Disclosed are methods, wherein the denaturing solution is 10×concentration, wherein the damaged DNA sample is mixed with thedenaturing solution to create a 1× concentration, and/or wherein thealkaline conditions comprise 15 to 50 mM NaOH.

Disclosed are methods, wherein the damaged DNA in the damaged DNA sampleis substantially denatured without further damaging the DNA, wherein thedamaged DNA sample is heated to a temperature of about 70° C. or lessand for a length of time of about 5 minutes or less, wherein thetemperature is about 60° C. to about 70° C., wherein the temperature isabout 50° C. to about 60° C., wherein the temperature is about 40° C. toabout 50° C., wherein the temperature is about 25° C. to about 40° C.,wherein the temperature is about 60° C. to about 70° C. and the lengthof time is about 3 minutes, and/or wherein the temperature is about 25°C. to about 50° C. and the length of time is about 5 minutes or more.

Disclosed are methods wherein altering the conditions comprises reducingthe pH of and cooling the denatured damaged DNA sample, wherein thetemperature to which the damaged DNA sample is heated is maintainedduring reduction of the pH of the denatured damaged DNA sample, whereinthe temperature to which the damaged DNA sample is heated is reducedbefore reduction of the pH of the denatured damaged DNA sample, and/orwherein the temperature to which the damaged DNA sample is heated isreduced during reduction of the pH of the denatured damaged DNA sample.

Disclosed are methods, wherein cooling the denatured damaged DNA sampleis commenced during reduction of the pH of the denatured damaged DNAsample, wherein cooling the denatured damaged DNA sample is commencedwhen the pH of the denatured damaged DNA sample is reduced.

Disclosed are methods, wherein the pH of the denatured damaged DNAsample is reduced by mixing the denatured damaged DNA sample with astabilization solution.

Disclosed are methods, wherein the pH of the denatured damaged DNAsample is reduced to the range of about pH 7.5 to about pH 8.0.

Disclosed are methods, wherein the pH of the denatured damaged DNAsample is reduced by mixing the denatured damaged DNA sample with astabilization solution.

Disclosed are methods, wherein the stabilization solution comprises abuffer, and/or wherein the buffer is phosphate buffer, Good buffer, BES,BICINE, CAPS, EPPS, HEPES, MES, MOPS, PIPES, TAPS, TES, TRICINE, sodiumcacodylate, sodium citrate, triethylammonium acetate, triethylammoniumbicarbonate, Tris, Bis-tris, or Bis-tris propane.

Disclosed are methods, wherein the stabilization solution comprises 800mM Tris-HCl, pH 4.1.

Disclosed are methods, wherein the stabilization solution comprises anacid.

Disclosed are methods, wherein the acid is hydrochloric acid, sulfuricacid, phosphoric acid, acetic acid, acetylsalicylic acid, ascorbic acid,carbonic acid, citric acid, formic acid, nitric acid, perchloric acid,HF, HBr, HI, H₂S, HCN, HSCN, HClO, monochloroacetic acid, dichloroaceticacid, trichloroacetic acid, or a carboxylic acid.

Disclosed are methods, wherein the carboxylic acid is ethanoic,propanoic, or butanoic.

Disclosed are methods, wherein the stabilization solution comprises aplurality of acidic agents.

Disclosed are methods, wherein the pH of the denatured damaged DNAsample is reduced to the range of about pH 9.0 to about pH 6.8, about pH9.0 to about pH 7.5, about pH 9.0 to about pH 8.0, about pH 8.5 to aboutpH 6.8, about pH 8.5 to about pH 7.5, about pH 8.5 to about pH 8.0,about pH 8.0 to about pH 6.8, about pH 8.0 to about pH 7.5.

Disclosed are methods, wherein the pH of the denatured damaged DNAsample is reduced to about pH 9.0 or less, about pH 8.5 or less, aboutpH 8.0 or less, about pH 7.5 or less.

Disclosed are methods, wherein the stabilization solution comprises oneor more salts.

Disclosed are methods, wherein the salt is Tris-HCl, Tris-EDTA, sodiumchloride, potassium chloride, magnesium chloride, sodium acetate,potassium acetate, magnesium acetate, or a combination.

Disclosed are methods, wherein the Tris-HCl is from pH 7.0 to 8.0.

Disclosed are methods, wherein the salt is Tris-EDTA.

Disclosed are methods, wherein the stabilization solution comprisesabout 50 mM to about 500 mM Tris and about 1 mM to about 5 mM EDTA.

Disclosed are methods, wherein the damaged DNA mixture is cooled at arate of about 1° C. per minute or less.

Disclosed are methods, wherein the damaged DNA mixture is cooled at arate of about 1% per minute or less.

Disclosed are methods, wherein the damaged DNA mixture is cooled to roomtemperature or lower from 60° C. to 70° C., from 50 to 60° C., from 40°C. to 50° C., 30° C. to 40° C., 50° C. to 70° C.

Disclosed are methods, wherein the denaturing solution comprises one ormore salts.

Disclosed are methods, wherein the salt is Tris-HCl, Tris-EDTA, sodiumchloride, potassium chloride, magnesium chloride, sodium acetate,potassium acetate, magnesium acetate, or a combination.

Disclosed are methods, wherein the Tris-HCl is from pH 7.0 to 8.0.

Disclosed are methods, wherein the salt is Tris-EDTA.

Disclosed are methods, wherein the denaturing solution comprises about50 mM to about 500 mM Tris and about 1 mM to about 5 mM EDTA.

Disclosed are methods, wherein the damaged DNA sample is comprised ofdegraded DNA fragments of genomic DNA.

Disclosed are methods, wherein replication and repair of the damaged DNAis accomplished by incubating the damaged DNA in the presence of a DNApolymerase.

Disclosed are methods, wherein the polymerase is a DNA polymerase thatcan extend the 3′-ends of the damaged DNA.

Disclosed are methods, wherein the DNA polymerase is φ29 DNA polymerase,BST DNA polymerase, Taq DNA polymerase, a modified form of Taq DNApolymerase, a Reverse Transcriptase, T4 DNA polymerase, T7 DNApolymerase, Pol I DNA polymerase, or a modified form of DNA PolymeraseI.

Disclosed are methods, wherein the DNA polymerase is φ29 DNA Polymerase.

Disclosed are methods, wherein the damaged DNA is amplified using a kit,wherein the kit comprises a denaturing solution, a stabilizationsolution, a set of primers, and a DNA polymerase.

Disclosed are methods, wherein the damaged DNA sample is a cell lysate,wherein the cell lysate is produced by exposing cells to alkalinecondition, wherein the cell lysate comprises a whole genome.

Disclosed are methods, comprising following exposure of the cells toalkaline conditions, exposing a first portion of the cell lysate toconditions that promote substantial denaturation of damaged DNA in thefirst portion of the cell lysate, wherein reducing the pH of the celllysate comprises reducing the pH of the first portion of the cell lysateto form a first stabilized cell lysate and reducing the pH of a secondportion of the cell lysate to form a second stabilized cell lysate,following reducing the pH of the cell lysate, mixing the secondstabilized cell lysate with the first stabilized cell lysate underconditions that promote transient denaturation of the ends of damagedDNA in the second stabilized cell lysate and that maintain substantialdenaturation of the damaged DNA in the first stabilized cell lysate,thereby forming a stabilized cell lysate mixture, and prior toincubating the stabilized cell lysate, cooling the stabilized celllysate mixture under conditions that promote annealing of the ends ofthe transiently denatured damaged DNA to the substantially denatureddamaged DNA, wherein incubating the stabilized cell lysate underconditions that promote replication of the genome also promotesreplication of the damaged DNA, wherein the annealed ends of the damagedDNA prime replication, wherein replication of the damaged DNA results inrepair of the replicated strands.

Disclosed are methods of amplifying damaged DNA, the method comprisingexposing a first damaged DNA sample to conditions that promotesubstantial denaturation of damaged DNA in the first damaged DNA sample,thereby forming a denatured damaged DNA sample, reducing the pH of thedenatured damaged DNA sample to form a stabilized denatured damaged DNAsample, mixing a second damaged DNA sample with the stabilized denatureddamaged DNA sample under conditions that promote transient denaturationof the ends of damaged DNA in the second sample and that maintainsubstantial denaturation of the damaged DNA in the stabilized denatureddamaged DNA sample, thereby forming a damaged DNA mixture, cooling thedamaged DNA mixture under conditions that promote annealing of the endsof the transiently denatured damaged DNA to the substantially denatureddamaged DNA, incubating the annealed damaged DNA under conditions thatpromote replication of the damaged DNA, wherein the annealed ends of thedamaged DNA prime replication, wherein replication of the damaged DNAresults in repair of the replicated strands, wherein during replicationat least one of the replicated strands is displaced by stranddisplacement replication of another replicated strand.

Disclosed are methods, wherein the temperature to which the firstdamaged DNA sample is heated is maintained during mixing of the seconddamaged DNA sample with the stabilized denatured damaged DNA sample.

Disclosed are methods, wherein the pH of the stabilized denatureddamaged DNA sample is not high enough nor low enough to cause furthersubstantial denaturation upon mixing second damaged DNA sample with thestabilized denatured damaged DNA sample.

Disclosed are methods, wherein the first damaged DNA sample is a portionof a damaged DNA sample, wherein the second damaged DNA sample is aportion of the same damaged DNA sample.

Disclosed are methods, wherein the first damaged DNA sample is from thesame source as the second damaged DNA sample.

Disclosed are methods, wherein the first damaged DNA sample is from thesame organism as the second damaged DNA sample.

Disclosed are methods, wherein the first damaged DNA sample is from thesame tissue as the second damaged DNA sample.

Disclosed are methods, wherein the second damaged DNA sample is mixedwith the stabilized denatured damaged DNA sample at a temperature andfor a length of time that transiently denatures the damaged DNA in thesecond damaged DNA sample.

Disclosed are methods, wherein the temperature is about 70° C. or lessand the length of time is about 30 seconds or less.

Disclosed are methods, wherein the second damaged DNA sample is mixedwith the stabilized denatured damaged DNA sample at a temperature thatdoes not further damaging the DNA.

Disclosed are methods, wherein the temperature is about 60° C. to about70° C., about 50° C. to about 60° C., about 40° C. to about 50° C.,about 25° C. to about 40° C., about 25° C. to about 70° C. Disclosed aremethods wherein the length of time is about 30 seconds.

EXAMPLES A. Example 1 Whole Genome Amplification using NucleaseResistant Hexamer Primers

This example describes a demonstration of an embodiment of the disclosedmethod and analysis and comparison of the results. The exemplifiedmethod is the disclosed multiple displacement amplification form ofwhole genome amplification using nuclease resistant random hexamerprimers. Some reactions in this example were performed withoutsubjecting the sample to denaturing conditions, a preferred form of thedisclosed method. In other reactions, the template DNA was subjected todenaturation prior to amplification. MDA was performed using φ29 DNApolymerase.

1. Materials and Methods

i. DNA and Enzymes.

A panel of human genomic DNA samples, the Human VariationPanel-Caucasian Panel of 100 (reference number HD100CAU) was obtainedfrom Coriell Cell Repositories. Human genomic DNA was also obtained fromPromega Corp. Thiophosphate-modified random hexamer(5′-NpNpNpNp^(s)Np^(s)N-3′) was synthesized at Molecular Staging, φ29DNA polymerase was from Amersham Pharmacia Biotech, and yeastpyrophosphatase was from Boehringer-Mannheim. DNA size markers (100 bpDNA ladder, 1 kb DNA ladder) were from Gibco BRL.

ii. Amplification of Human Genomic DNA.

Human genomic DNA (300 ng to 0.03 ng, as indicated) was placed into 0.2ml tubes in a total volume of 50 μl, yielding final concentrations of 25mM Tris-HCl, pH 7.5, 50 mM KCl, 10 mM MgCl₂, and 100 μMexonuclease-resistant hexamer. A heat-treatment step (that is, exposureto denaturing conditions) to increase primer annealing was included oromitted, as indicated, for individual experiments. Annealing reactionswere heated to 95° C. for 3 minutes and chilled to 4° C. in a PCR SystemThermocycler (Perkin Elmer). Reactions were then brought to a finalvolume of 100 μl, containing final concentrations of 37 mM Tris-HCl, pH8.0, 50 mM KCl, 10 mM MgCl₂, 5 mM (NH₄)₂SO₄, 1.0 mM dNTPs, 1 unit/ml ofyeast pyrophosphatase, 50 μM exonuclease-resistant hexamer, and 800units/ml φ29 DNA polymerase. Radioactively labeled α-[³²P] dCTP,approximately 60 cpm/pmol total dNTPs, was added as indicated. Reactionswere incubated for 18 hours at 30° C. Incorporation of acid-precipitableradioactive deoxyribonucleotide product was determined with glass fiberfilters. After the reactions were terminated, 3 μl aliquots were cleavedwith restriction endonuclease AluI and analyzed by electrophoresisthrough a 1.0% agarose gel in Tris-borate-EDTA buffer, stained withGelStar (Molecular Probes) or SYBR Green (Molecular Probes), and imagedwith a Storm 860 PhosphorImager (APB). Denaturing gel analysis wascarried out by electrophoresis through a 1.0% agarose gel in 30 mM NaOH,1 mM EDTA. The radioactive products in the dried gel were visualizedwith the Storm 860 PhosphorImager.

iii. Southern Analysis.

10 μg of whole genome amplified DNA or human genomic DNA controls weredigested with EcoRI restriction endonuclease and separated through a 1%agarose gel in 1×TBE buffer. Standard Southern analysis procedure(Southern, Detection of specific sequences among DNA fragments separatedby gel electrophoresis. J Mol Biol. 98:503–517 (1975)) was performedusing a Hybond-N+ membrane (Amersham Pharmacia Biotech, Piscataway,N.J.). An exon fragment probe of parathyroid hormone (p20.36) and RFLPmarker probes for the D13S12 (p9D11) and Thyroglobulin (pCHT. 16/8) lociwere obtained from American Type Culture Collection. Probes wereradiolabeled using the NEBlot random primer labeling method (New EnglandBiolabs, Beverly, Mass.). The membrane was prehybridized for 1 hr andhybridized to the radiolabeled probe overnight in a MembraneHybridization Buffer (Amersham Pharmacia Biotech, Piscataway, N.J.). Thehybridized membrane was washed in 2×SSC and 0.1% SDS twice for 5 min atroom temperature, 1×SSC and 0.1% SDS for 15 min at 42° C., and 0.1×SSCtwice for 15 min at 65° C. The membrane was then exposed overnight andanalyzed using the Storm 860 PhosphorImager.

iv. Quantitative PCR Analysis.

TaqMan analysis was performed using the ABI 7700 according to themanufacturer's specifications (Applied Biosystems, Foster City, Calif.)using 1 μg of amplified DNA as template. TaqMan assay reagents for the 8loci tested were obtained from ABI. The 8 loci and their chromosomeassignments were, acidic ribosomal protein (1p36.13); connexin 40(1q21.1); chemokine (C—C motif) receptor 1 (3p21); chemokine (C—C motif)receptor 6 (6q27); chemokine (C—C) receptor 7 (17q21); CXCR5 Burkittlymphoma receptor 1 (chr. 11); c-Jun (1p32-p31); and MKP1 dualspecificity phosphatase 1 (5q34). Connexin 40 is located near thecentromere and chemokine (C—C motif) receptor 6 is located near thetelomere. A standard curve for input template was generated to determinethe loci copy number in amplified DNA relative to that of genomic DNA.The standard curve was generated from 0, 0.001, 0.01, 0.1, 0.5, and 1 μgof genomic DNA.

v. Amplification of Human Genomic DNA by Degenerate Oligonucleotide PCR(DOP-PCR).

Human genomic DNA (ranging from 300 ng to 0.03 ng) was amplified asdescribed (Telenius et al., Genomics. 13:718–725 (1992)). Radioactivelylabeled α-[³²P] dCTP, approximately 60 cpm/pmol total dNTPs, was addedto the reaction for the quantitation of PCR product yield. Taq DNAPolymerase was from Invitrogen Life Technologies, Carlsbad, Calif.DOP-PCR amplifications were carried out using a GeneAmp 9700 PCR Systemthermocycler (Applied Biosystems, Foster City, Calif.). Locusrepresentation in the DOP-PCR product was quantitatively analysed usingthe TaqMan assay (Invitrogen Life Technologies, Carlsbad, Calif.).

vi. Amplification of Human Genomic DNA by Primer ExtensionPreamplification (PEP).

Human genomic DNA (ranging from 300 ng to 0.03 ng) was placed into 0.2ml tubes in a total volume of 60 μl, yielding final concentrations of 33μM PEP random primer (5′-NNN NNN NNN NNN NNN-3′) as described (Zhang etal., Whole genome amplification from a single cell: implications forgenetic analysis. Proc Natl Acad Sci USA. 89:5847–5851 (1992)).Radioactively labeled α-[³²P] dCTP, approximately 60 cpm/pmol totaldNTPs, was added to the reaction for the quantitation of PCR product.PEP reactions were carried out using a GeneAmp 9700 PCR Systemthermocycler (Applied Biosystems, Foster City, Calif.). Locusrepresentation in the PEP product was quantitatively analysed using theTaqMan assay (Invitrogen Life Technologies, Carlsbad, Calif.).

vii. Genotyping of Single Nucleotide Polymorphisms.

The SNPs analyzed here had the following chromosomal locations; 1822,251, and 221, 13q32; 465, 458, and 474, 19q13; VCAM, 1p31; IL-8, 4q13;PDK2–2, 17p; SNP21, not known. Assays were carried out as described byFaruqi et al., High-Throughput Genotyping of Single NucleotidePolymorphisms with Rolling Circle Amplification. BMC Genomics, 2:4(2001). Briefly, DNA denaturation, annealing and ligation reactions werecarried out in an Eppendorf Master Cycler (Eppendorf Scientific,Germany). Exponential RCA reactions were performed in the Real-Time ABI7700 Sequence Detector (Perkin Elmer). Two controls lacking ligase werealso carried out for each SNP, confirming the specificity of the assays.The DNA samples were digested with the restriction endonuclease AluIbefore being used as template in the ligation reaction. Ligationreactions were set up in 96-well MicroAmp Optical plates (Perkin Elmer)in a 10 μl reaction volume containing 1 unit Ampligase (EpicentreTechnologies), 20 mM Tris-HCl (pH 8.3), 25 mM KCl, 10 mM MgCl₂, 0.5 mMNAD, and 0.01% Triton® X-100. Standard reactions contained 0.5 pM opencircle padlock and 100 ng of Alu I digested genomic DNA. DNA wasdenatured by heating the reactions at 95° C. for 3 min followed byannealing and ligation at 60° C. for 20 min. 20 μl of ERCA mix was addedto the 10 μl ligation reaction. The ERCA mix contained 5% TMA oxalate,400 μM dNTP mix, 1 μM each of the two primers, 8 units of Bst polymerase(New England Biolabs, Mass.), and 1× modified ThermoPol II reactionbuffer containing 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH₄)₂SO₄and 0.1% Triton®X-100.

viii. Comparative Genome Hybridization.

Genomic DNA preparations were nick-translated to incorporate nucleotidesmodified with biotin for amplified samples or digoxigenin forunamplified control samples. Equimolar amounts of amplified andunamplified DNA were co-hybridized in the presence or absence of CotIDNA to suppress repetitive DNA cross-hybridization. Specifichybridization signals were detected by avidin-FITC and anti-digoxigeninrhodamine. Captured images of metaphase chromosomes were analyzed usingthe Applied Imaging CGH software program and fluorescence profiles weregenerated. As controls, differentially labeled amplified or unamplifiedDNAs were mixed, hybridized, detected and subjected to ratio analysis asoutlined above.

2. Results

Using the embodiment of the disclosed method, 30 pg (approximately 9genomic copies) of human genomic DNA was amplified to approximately 30μg within 4 hours. The average fragment length was greater than 10 kb.The amplified human DNA exhibited normal representation for 10 singlenucleotide polymorphisms (SNPs). Maximum bias among 8 chromosomal lociwas less than 3-fold in contrast to four to six orders of magnitude forPCR-based WGA methods. Human DNA amplified with the disclosed method isuseful for several common methods of genetic analysis, includinggenotyping of single nucleotide polymorphisms (SNP), chromosomepainting, Southern blotting and RFLP analysis, subcloning, and DNAsequencing.

It has been discovered that the use of random hexamer primers and φ29DNA polymerase in multiple displacement amplification, a cascading,strand displacement reaction (U.S. Pat. No. 6,124,120 to Lizardi), willreadily amplify linear, human genomic DNA. Amplification of genomic DNAby the disclosed form of MDA at 30° C. is exponential for 4–6 hours. Theeffect of template concentration on amplification yield in the disclosedmethod is shown in FIG. 1. 100 fg to 10 ng human genomic DNA wasdenatured at 95° C. for 5 min, and then MDA was carried out at 30° C. asdescribed above. Aliquots were taken at the times indicated in FIG. 1 toquantitate DNA synthesis. Amplification reactions (100 μl) yieldedapproximately 25–30 μg DNA product regardless of the starting amount ofgenomic DNA over a 5-log range (100 fg to 10 ng; FIG. 1). For someapplications, this allows subsequent genetic analysis without the needto measure or adjust DNA concentration.

The products of the MDA reaction were characterized as follows.Radioactively labeled human genome amplification samples (0.6 μg) wereelectrophoresed through an alkaline agarose gel (1%, Tris-Borate EDTAbuffer), stained, and imaged as described above. Average product lengthexceeded 10 kb.

To examine the integrity of amplified DNA, a restriction fragment withinthe human parathyroid hormone gene (chromosome 11p15.2–15.1) wasanalyzed on Southern blots. A 1.9 kb restriction enzyme fragment wasobserved for MDA-based WGA products amplified from as few as 10 genomiccopies (or 10⁶-fold amplification). These MDA reactions included a heatdenaturation step and amplification was carried out as described above.EcoRI DNA digests were probed using a radioactively-labeled genomicfragment of the parathyroid hormone gene (p20.36) that hybridized to anapproximately 1.9 kb DNA fragment. The EcoRI-cleaved DNA preparationswere genomic DNA, DNA amplified by MDA from varying amounts of inputgenomic DNA, or an MDA reaction that lacked input genomic DNA template.This demonstrates that the products of MDA are long enough to yieldspecific DNA fragments several kb in length after cleavage byrestriction endonucleases.

While PCR-based WGA methods typically generate products of only severalhundred nucleotides in length (Telenius et al., Genomics. 13:718–725(1992); Cheung and Nelson, Proc Natl Acad Sci USA. 93:14676–14679(1996); Zhang et al., Proc Natl Acad Sci USA. 89:5847–5851 (1992)),products of the disclosed method were of sufficient length and integrityfor RFLP-based genotyping: 16 random individuals were correctlygenotyped by the presence of a 2.1 kb, and 1.1 kb Pst I fragment.Specifically, PstI DNA digests were probed using a radioactively-labeledgenomic fragment of the RFLP marker D113S12 locus (p9D11) thathybridized to an invariant 3.8 kb DNA fragment and a polymorphic 2.1 kb(allele A) or 1.1 kb (allele B) DNA fragment. The PstI-cleaved DNApreparations were genomic DNA and 5 different patient DNAs amplified byMDA without any heat denaturation of the DNA template (10,000×amplification).

Omission of denaturation conditions prior to MDA was useful fordetection of restriction fragments greater than 5 kb in length. MDAreactions were performed with or without heat denaturation of thegenomic target DNA heterozygous for two thyroglobulin alleles.Amplification was carried out as described above. TaqI DNA digests wereprobed using a radioactively-labeled genomic fragment of thethyroglobulin gene (pCHT. 16/8) that hybridized to invariant 1 kb and 3kb DNA fragments and a polymorphic 5.8 kb (allele A) or 5.2 kb (alleleB) DNA fragment. The TaqI-cleaved DNA preparations were genomic DNA, DNAamplified by MDA reaction (10,000×) with a 95° C. preheating step, andan MDA reaction (10,000×) without the preheating step. MDA without heatdenaturation gave a good yield of DNA fragments 5.2 and 5.8 kb in size,while neither of the 5.2 or 5.8 kb DNA fragments were visible using MDAwith heat denaturation of the template.

The most useful results from whole genome amplification are obtainedwhen the amplification provides complete coverage of genomic sequencesand minimal amplification bias. It is also preferred that theamplification product perform similarly to unamplified genomic DNAduring subsequent genetic analysis. Genome coverage after MDA with heatdenaturation of the template was examined for 10 randomly distributedSNPs after amplifications of 100-, 10,000-, or 100,000-fold. Thepresence of all loci was confirmed in the amplified DNA with theexception of one locus (PDK2-2) in 100,000-fold amplified DNA (Table 1).MDA DNA from 72 individuals was genotyped for one of these SNPs.Following 100-fold amplification by MDA, genotyping accuracy was 97% (70of 72 genotypes scored correctly, Table 1, SNP 1822), a result that wasindistinguishable from unamplified genomic DNA genotyped by the samemethod. MDA-based WGA thus offers an attractive alternative to multiplelocus-specific PCR preamplifications for large SNP scoring studies,especially where sample availability is limited.

TABLE 1 Fold whole genome amplification SNP 100X 10,000X 100,000Xdesignation Correct SNP calls/total assays 1822 70/72 4/4 3/4  251 8/84/4 4/4  221 3/4 3/4 4/4  465 4/4 3/4 3/4  458 4/4 3/4 3/4  474 4/4 3/44/4 VCAM 4/4 4/4 4/4 IL-8 4/4 4/4 3/4 SNP21 4/4 4/4 3/4 PDK2-2 4/4 4/4 0/12

Sequence bias can occur in amplification methods, and may result fromfactors such as priming efficiency, template accessibility, GC content,and proximity to telomeres and centromeres. Amplification bias of thedisclosed MDA method was examined by TaqMan quantitative PCR for 8genes, including one near the centromere of chromosome I (connexin 40)and one adjacent to the telomere of chromosome 6 (chemokine (C—C motif)receptor 6). For 100, 1,000, and 10,000-fold MDA without heatdenaturation, the maximum amplification biases were only 2.7, 2.3, and2.8 respectively, expressed as the ratio of the most highly representedgene to the least represented gene. In contrast, two WGA methods basedon PCR, DOP-PCR (Telenius et al., Genomics. 13:718–725 (1992); Cheungand Nelson, Proc Natl Acad Sci USA. 93:14676–14679 (1996)) and PEP(Zhang et al., Proc Natl Acad Sci US A. 89:5847–5851 (1992)), exhibitedamplification bias of 4–6 orders of magnitude. These values wereconsistent with literature values for the bias of PCR-mediated WGAmethods; PEP has been reported to generate an amplification bias of upto 50-fold even between two alleles of the same gene (Paunio et al.,Clin. Chem. 42:1382–1390 (1996)). Significantly, the 3-foldamplification bias of MDA remained almost constant between 100 and100,000-fold amplification. An absence of significant sequence biasobserved in MDA-based WGA may be explained by the extraordinaryprocessivity of Φ29 DNA polymerase (Blanco et al., J. Biol. Chem.264:8935–8940 (1989)). Tight binding of the polymerase to the templateassures replication through obstacles caused by DNA primary or secondarystructure.

Surprisingly, TaqMan quantification indicated that certain gene lociwere enriched by MDA in amplified DNA; locus representation was >100% ofthe representation in genomic DNA. Representation of mitochondrial DNAwas the same between starting gDNA and amplified DNA. Southern blots andchromosome painting experiments indicated that repetitive sequences wereunderrepresented in MDA product, conferring an effective enrichment ofgenes. Thus, amplified DNA contained between 100–300% copy numbers of 8genes relative to genomic DNA. Additional studies will be necessary toidentify the extent and type of repetitive element under-representation;one hypothesis for this observation is that primers corresponding tohighly repetitive elements become depleted during MDA. However, incontrast to PCR-based WGA products, which contain up to 70%amplification artifacts (Cheung and Nelson, Whole genome amplificationusing a degenerate oligonucleotide primer allows hundreds of genotypesto be performed on less than one nanogram of genomic DNA. Proc Natl AcadSci USA. 93:14676–14679 (1996)), MDA-based WGA products appear to beentirely derived from genomic sequences.

Whole genome MDA was also tested for uniformity of chromosome coverageby comparative genomic hybridization. MDA-generated DNA was cohybridizedto metaphase chromosomes with an equivalent amount of unamplifiedgenomic DNA. Amplification reactions included a heat denaturation stepand amplification was carried out as described above. Amplified andunamplified DNA samples were nick-translated to incorporate biotinnucleotide and digoxigenin nucleotide, respectively. Specific signalswere detected by avidin FITC and anti-digoxigenin rhodamine. With CotIsuppression, the hybridization patterns of MDA probes and unamplifiedprobes were indistinguishable even after 100,000-fold amplification.Without CotI suppression, however, the 100,000-fold amplified probe gavereduced centromeric signals, indicating some loss of repetitivecentromeric sequences. The uniformity of signal along the length of thechromosome arms was further evidence that MDA-based WGA does not inducesignificant amplification bias. These results indicated that MDAcompared favorably with DOP PCR for preparation of chromosome paintingprobes (Kim et al., Whole genome amplification and molecular geneticanalysis of DNA from paraffin-embedded prostate adenocarcinoma tumortissue. J Urol. 162:1512–1518 (1999); Klein et al., Comparative genomichybridization, loss of heterozygosity, and DNA sequence analysis ofsingle cells. Proc Natl Acad Sci USA 96:4494–4499 (1999); Wells et al.,Detailed chromosomal and molecular genetic analysis of single cells bywhole genome amplification and comparative genomic hybridization.Nucleic Acids Res 27:1214–1218 (1999)), but that unlike the latter,suppression hybridization may be unnecessary for detection of singlecopy sequences. This method should be invaluable for DNA probepreparation for comparative genome hybridization, karyotyping andchip-based genetic analysis from limited patient DNA sources such asneedle biopsy material or amniocentesis samples.

For genome subcloning and sequencing MDA appears to have severalintrinsic advantages over PCR-based methods. Product size of >10 kb iscompatible with genome subcloning. Since no in vivo propagation ofamplified material is necessary, MDA may represent an efficient methodfor amplifying “poisonous” genomic sequences. In addition, φ29 DNApolymerase used for MDA has an error rate of 1 in 10⁶–10⁷ (Esteban etal., Fidelity of Phi29 DNA polymerase. Comparison between protein-primedinitiation and DNA polymerization. J. Biol. Chem. 268:2719–2726 (1993))in contrast to approximately 3 in 10⁴ for PCR with Taq DNA polymerase(Eckert and Kunkel, DNA polymerase fidelity and the polymerase chainreaction. PCR Methods and Applications. 1:17–24 (1991)). Therefore, PCRaccumulates about one mutation per 900 bases (Saiki et al.,Primer-directed enzymatic amplification of DNA with a thermostable DNApolymerase. Science 239:487–491 (1988)) after 20 cycles. Sequence errorrates of cloned MDA-based WGA products appear to be similar to those forcloned genomic DNA. Furthermore, minimal amplification bias, uniformyields, and assay simplicity make MDA amenable to the automated,high-density microwell formats used for genome subcloning andsequencing.

In summary, the utility of MDA-based WGA was demonstrated for a varietyof uses including quantitative PCR, SNP genotyping, Southern blotanalysis of restriction fragments, and chromosome painting. Severalsituations may be contemplated where MDA may represent the method ofchoice for WGA: Firstly, applications where faithful replication duringWGA is necessary, such as molecular cloning, or single cell analysis;Secondly, applications where adequate genome representation during WGAis critical, such as genome-wide SNP genotyping studies; And thirdly,where minimization of bias during WGA is important, particularlycytogenetic testing such as pre-natal diagnosis (Harper and Wells,Recent advances and future developments in PGD. Prenat Diagn.19:1193–1199 (1999)), comparative genome hybridization (Wells andDelhanty, Comprehensive chromosomal analysis of human preimplantationembryos using whole genome amplification and single cell comparativegenomic hybridization. Mol Hum Reprod. 6:1055–1062 (2000)), andassessment of loss of heterozygosity (Paulson et al., Loss ofheterozygosity analysis using whole genome amplification, cell sorting,and fluorescence-based PCR. Genome Res. 9:482–491 (1999)). Additionalsituations where there is a significant need for WGA includeamplification of DNA from micro-dissected tissues (Kim et al., Wholegenome amplification and molecular genetic analysis of DNA fromparaffin-embedded prostate adenocarcinoma tumor tissue. J Urol.162:1512–1518 (1999)), buccal smears (Gillespie et al., HLA class IItyping of whole genome amplified mouth swab DNA. Tissue Antigens.56:530–538 (2000)), and archival, anthropological samples (Buchanan etal., Long DOP-PCR of rare archival anthropological samples. Hum Biol.72:911–925 (2000)). Finally, DNA amplified from sorted, individualchromosomes may be used for the generation of whole chromosome-specificpainting probes (Guillier-Gencik et al., Generation of whole-chromosomepainting probes specific to each chicken macrochromosome. Cytogenet CellGenet. 87:282–285 (1999)).

B. Example 2 Increasing Time of Incubation at 95° C. Causes IncreasingTemplate DNA Strand Breakage

This example demonstrates that significant template DNA strand breakageis generated by incubation at 95° C. (which is used in typicalamplification reactions to denature the DNA), and that strand breakageis reduced by decreasing the duration of heat treatment. As with mostnucleic acid amplification techniques, the integrity of the starting DNAtemplate can have an important effect on the rate and yield of theamplified product. In reactions where the nucleic acid to be amplifiedis degraded, the yield of amplified product may be reduced both inquality and quantity. This example demonstrates the reduction oftemplate DNA strand breakage by decreasing time of incubation at 95° C.

Six reactions were carried out under the conditions used for DNAtemplate strand separation with heat-denaturation in order to illustratethe degradation of template DNA. Human genomic DNA (10 μg) was placedinto a 0.2 ml tube in a total volume of 50 μl, yielding finalconcentrations of 25 mM Tris-HCl, pH 7.5, 50 mM KCl, 10 mM MgCl₂.Reactions were heated to 95° C. in a PCR System Thermocycler (PerkinElmer), and an aliquot of 8 μl was taken and put on ice at indicatedtimes. For each time point, a 2 μl aliquot was analyzed byelectrophoresis through a 0.8% alkaline agarose gel (30 mM NaOH, 1 mMEDTA). After electrophoresis, the gel was neutralized with 1×TBE,stained with SYBR Green II (Molecular Probes), and imaged with a Storm860 PhosphorImager (APB). The total amount of DNA imaged was determinedfor each lane of the gel and the fragment size at which 50% of the DNAwas larger, and 50% was smaller, was determined for the samples drawn ateach time point. The results are shown in FIG. 2.

As can be seen, significant breakage of template DNA occurs afterincubation at 95° C. for longer than three minutes. Such DNA breakage issubstantially reduced when incubation is limited to one minute.

C. Example 3 Increasing Time of DNA Template Incubation at 95° C. CausesDecreased Rate and Yield of DNA Amplification

This example demonstrates that increased time of template DNA incubationat 95° C. (which is used in typical amplification reactions to denaturethe DNA) causes a reduction in both the rate and the yield with whichDNA is amplified by MDA. Omission of DNA template incubation at 95° C.results in the greatest rate and yield of DNA amplification.

Six MDA reactions were carried out using template DNA treated at 95° C.under the conditions described in Example 2. Purified human genomic DNA(3 ng) was placed into 0.2 ml tubes in a total volume of 50 μl,containing 25 mM Tris-HCl, pH 7.5, 50 mM KCl, and 10 mM MgCl₂. Reactionswere heated to 95° C. for the time indicated and chilled to 4° C. in aPCR System Thermocycler (Perkin Elmer). These reactions were thenbrought to a final volume of 100 μl, containing final concentrations of37 mM Tris-HCl, pH 7.5, 50 mM KCl, 10 mM MgCl₂, 50 mMexonuclease-resistant hexamer, 5 mM (NH₄)₂SO₄, 1.0 mM dNTPs, 1 unit/mlof yeast pyrophosphatase, and 800 units/ml φ29 DNA polymerase.Radioactively labeled α-[³²P] dCTP, approximately 60 cpm/pmol totaldNTPs, was added and reactions were incubated for 22 hours at 30° C.Aliquots were taken at the times indicated and incorporation ofacid-precipitable radioactive deoxyribonucleotide product was determinedwith glass fiber filters. The results are shown in FIG. 3.

As can be seen, omission of heat treatment of the DNA template resultsin the optimal rate and yield of DNA synthesis (see 0 min curve).Increasing duration of DNA template heat treatment resulted inprogressively reduced rate and yield of DNA synthesis (see 1 min, 3 min,5 min, 10 min, and 20 min curves).

D. Example 4 Increasing Time of DNA Template Incubation at 95° C.Results in Decreasing DNA Product Strand Size

This example demonstrates that increased time of template DNA incubationat 95° C. (which is used in typical amplification reactions to denaturethe DNA) causes a reduction in the length of the DNA products amplifiedby MDA. Omission of DNA template incubation at 95° C. results in thegreatest size of DNA amplification products.

Six MDA reactions were carried out using template DNA treated at 95° C.under the conditions described in Example 3. Radioactively labeledα-[³²P] dCTP was added, approximately 60 cpm/pmol total dNTPs. Reactionswere incubated for 22 hours at 30° C. and aliquots were taken at thetimes indicated. For each time point, a 2 μl aliquot was analyzed byelectrophoresis through a 0.8% alkaline agarose gel (30 mM NaOH, 1 mMEDTA). After electrophoresis, the gel was neutralized and imaged asdescribed in Example 2. The total amount of DNA imaged was determinedfor each lane of the gel and the fragment size at which 50% of the DNAwas larger, and 50% was smaller, was determined for the samples fromeach time point. The results are shown in FIG. 4.

As can be seen, omission of heat treatment of the DNA template resultsin the synthesis of the largest DNA products. Increasing duration of DNAtemplate heat treatment resulted in progressively reduced DNA productsize.

E. Example 5 Omission of DNA Template Incubation at 95° C. Results inIncreased Locus Representation in DNA Products Amplified by MDA

This example demonstrates that omission of template DNA incubation at95° C. (which is used in typical amplification reactions to denature theDNA) results in no loss in the representation of eight randomly selectedloci in DNA products amplified by MDA. Omission of DNA templateincubation at 95° C. actually results in an increase in locusrepresentation of DNA amplification products relative to templategenomic DNA.

Two MDA reactions were carried out using template DNA either treated ornot treated at 95° C. Purified human genomic DNA (3 ng) was placed intoa 0.2 ml tube in a total volume of 50 μl, containing 25 mM Tris-HCl, pH7.5, 50 mM KCl, 10 mM MgCl₂, and 100 μM exonuclease-resistant hexamer.The annealing reaction was heated to 95° C. for 3 minutes and chilled to4° C. in a PCR System Thermocycler (Perkin Elmer). The reaction was thenbrought to a final volume of 100 μl, containing final concentrations of37 mM Tris-HCl, pH 7.5, 50 mM KCl, 10 mM MgCl₂, 50 μMexonuclease-resistant hexamer, 5 mM (NH₄)₂SO₄, 1.0 mM dNTPs, 1 unit/mlof yeast pyrophosphatase, and 800 units/ml φ29 DNA polymerase. Foramplification lacking the heat denaturation step, DNA template (3 ng)was placed directly into a 0.2 ml tube in a total volume of 100 μlcontaining 37 mM Tris-HCl, pH 7.5, 50 mM KCl, 10 mM MgCl₂, and 50 μMexonuclease-resistant hexamer, 5 mM (NH₄)₂SO₄, 1.0 mM dNTPs, 1 unit/mlof yeast pyrophosphatase, and 800 units/ml φ29 DNA polymerase. Reactionswere incubated for 18 hours at 30° C.

TaqMan® quantitative PCR analysis was performed using the ABI 7700according to the manufacturer's specifications (Applied Biosystems,Foster City, Calif.) using 1 μg of MDA-amplified DNA as template.TaqMan® assay reagents for the 8 loci tested were obtained from ABI. The8 loci and their chromosome assignments were, acidic ribosomal protein(1p36.13); connexin 40 (1q21.1); c-Jun (1p32-p31); MKP1 dual specificityphosphatase 1 (5q34); chemokine (C—C motif) receptor 7 (17q21);chemokine (C—C motif) receptor 1 (3p21); CXCR5 Burkitt lymphoma receptor1 (chr. 11); and chemokine (C—C motif) receptor 6 (6q27). Connexin 40 islocated near the centromere and chemokine (C—C motif) receptor 6 islocated near the telomere. A standard curve for input template wasgenerated to determine the loci copy number in amplified DNA relative tothat of genomic DNA. The standard curve was generated from 0, 0.001,0.01, 0.1, 0.5, and 1 μg of genomic DNA. The locus representation wasexpressed as a percent, relative to the locus representation in theinput genomic DNA, and was calculated as the yield of quantitative PCRproduct from 1 μg amplified DNA divided by the yield from 1 μg genomicDNA control. The results are shown in FIG. 5.

As can be seen, there is no reduction of locus representation in DNAamplified from template DNA without heat treatment at 95° C. However,significant loss of locus representation was observed from template DNAheat-denatured for 3 min at 95° C.

F. Example 6 Amplification Bias of Loci Amplified by MDA isSignificantly Lower than Amplification Bias of DNA Amplified by PEP orDOP-PCR

This example demonstrates that, for 100-, 1,000-, and 10,000-foldMDA-amplified DNA, omission of template DNA incubation at 95° C. (whichis used in typical amplification reactions to denature the DNA) resultsin low bias in the representation of eight randomly selected loci in DNAproducts. In contrast, two whole genome amplification (WGA) methodsbased on PCR, DOP-PCR and PEP, exhibit amplification biases of 2–6orders of magnitude.

MDA reactions were carried out using template DNA not treated at 95° C.as described in Example 5. Reactions (100 μl) contained 300, 30, 3, or0.3 ng DNA, resulting in approximately 100-, 1000-, 10,000-, and100,000-fold DNA amplification.

Amplification of human genomic DNA by degenerate oligonucleotide PCR(DOP-PCR; Telenius et al., Genomics. 13:718–725 (1992); Cheung andNelson, Proc Natl Acad Sci USA. 93:14676–14679 (1996)) was carried outas follows. Human genomic DNA (ranging from 300 ng to 0.03 ng) wasplaced into 0.2 ml tubes in a total volume of 50 μl, yielding finalconcentrations of 2 μM DOP Primer (5′-CCG ACT CGA GNN NNN NAT GTG G-3′(SEQ ID NO:20); N=A, G, C, or T in approximately equal proportions), 200μM dNTPs, 10 mM Tris Cl (pH 8.3), 0.005% (v/v) BRIJ 35, 1.5 mM MgCl₂,and 50 mM KCl. Radioactively labeled α-[³²P] dCTP, approximately 60cpm/pmol total dNTPs was added to the reaction for quantitation of DNAsynthesis as described for MDA product quantitation in Example 3. Afterthe initial denaturation of the template at 95° C. for 5 min, 2.5 unitsTaq DNA Polymerase (Invitrogen Life Technologies, Carlsbad, Calif.) wasadded, followed by 5 cycles of 94° C. for 1 min, 30° C. for 1.5 min,ramping up to 72° C. in 3 min and elongation at 72° C. for 3 min, andthen 35 cycles of 94° C. for 1 min, 62° C. for 1 min, and 72° C. for 2min (+14 extra seconds/cycle). A final elongation was done at 72° C. for7 min. Amplification reactions were carried out using a GeneAmp 9700 PCRSystems Thermocycler (Applied Biosystems, Foster City, Calif.).

Amplification of human genomic DNA by primer extension preamplification(PEP; Zhang et al., Proc Natl Acad Sci USA. 89:5847–5851 (1992)) wascarried out as follows. Human genomic DNA (ranging from 300 ng to 0.03ng) was placed into 0.2 ml tubes in a total volume of 60 μl, yieldingfinal concentrations of 33 uM PEP random primer (5′-NNN NNN NNN NNNNNN-3′), 100 uM dNTPs, 10 mM Tris-HCl, pH 8.3 (20° C.), 1.5 mM MgCl₂, 50mM KCl, and 5 units of Taq DNA Polymerase (Invitrogen Life Technologies,Carlsbad, Calif.). Radioactively labeled α-[³²P] dCTP, approximately 60cpm/pmol total dNTPs was added to the reaction for quantitation of PCRproduct yield as described for MDA product quantitation in Example 3.The PEP reaction was performed for 50 cycles at 92° C. for 1 min, 37° C.for 2 min, ramping up to 55° C. at 10 sec/degree, and elongation at 55°C. for 4 min. PEP reactions were carried out using a GeneAmp 9700 PCRSystems Thermocycler (Applied Biosystems, Foster City, Calif.).

TaqMan® quantitative PCR analysis was performed as described in Example5, and maximum amplification bias between loci was calculated bydividing the high locus representation value by the low value for eachlevel of fold amplification. The results are shown in FIG. 6.

The relative representation of eight loci is depicted in FIG. 6 foramplification reactions carried out by three different WGA procedures.The X-axis represents the fold amplification in the amplified DNA usedas template for quantitative PCR; the Y-axis is the locusrepresentation, expressed as a percent, relative to input genomic DNA,which is calculated as the yield of quantitative PCR product from 1 μgamplified DNA divided by the yield from 1 μg genomic DNA control. Theresults for eight loci are indicated as follows; CXCR5, open diamonds;connexin40, open triangles; MKP1, open squares; CCR6, open circles;acidic ribosomal protein, filled diamonds; CCR1, filled triangles; cJUN,filled squares; CCR7, filled circles. FIG. 6A depicts the percentrepresentation for eight loci derived from MDA-amplified DNA. FIG. 6Bdepicts the percent representation for eight loci present in DNAamplified using DOP-PCR. FIG. 6C depicts the percent representation foreight loci present in PEP-amplified DNA.

As can be seen, for 100-, 1,000-, and 10,000-fold amplified MDA, themaximum amplification biases were only 2.7, 2.3, and 2.8 respectively,expressed as the ratio of the most highly represented gene to the leastrepresented gene. Significantly, the 3-fold amplification bias of MDAremained almost constant between 100- and 100,000-fold amplification(FIG. 6A). In contrast, amplification by the DOP-PCR method exhibited anamplification bias ranging between 4 and 6 orders of magnitude (FIG.6B). In addition, the PEP method exhibited an amplification biasspanning 2–4 orders of magnitude (FIG. 6C).

G. Example 7 Amplification using Nested Primers Yields SpecificAmplification of c-jun Sequences

This example demonstrates the amplification of a specific DNA regionfrom a complex mixture of DNA sequences using φ29 DNA polymerase withsequence-specific, nested primers.

Amplification reactions were carried out either with or without a heatdenaturing/annealing step using either exonuclease-resistant hexamers ora nested set of 19 exonuclease-resistant sequence-specific primers. Thenested primers used in this example are listed in Table 2. The presenceof an asterisk in the nucleotide sequence indicates the presence of aphosphorothioate bond. The nested primers are designed to hybridize toopposite strands on each side of the human c-jun gene, and the closestleft and right primers encompass a 3420 bp fragment containing the c-jungene. On each side of the c-jun gene, these nested primers are spaced150–400 nucleotides between each other. The region of human DNAencompassing the recognition sites for these primers can be accessedfrom Genbank using Accession Number AL136985 and spans positions 65001to 73010 of the nucleotide sequence.

TABLE 2 Left Primers (5′ to 3′) c-Jun TCC ATC ACG AGT TAT GC*A* C (SEQID NO:1) L9 c-Jun TGG AGT TAC TAA GGG AA*G* C (SEQ ID NO:2) L8 c-Jun ACTGAG TTC ATG AAC CC*T* C (SEQ ID NO:3) L7 c-Jun ATT AAC TCA TTG AAG GC*C*C (SEQ ID NO:4) L6 c-Jun TCT GTG CTG TAC TGT TG*T* C (SEQ ID NO:5) L5c-Jun AGT TTG GCA AAC TGG GC*T* C (SEQ ID NO:6) L4 c-Jun TGG CTC TTG GTATGA AA*A* G (SEQ ID NO:7) L3 c-Jun ACT GTT AGT TTC CAT AG*G* C (SEQ IDNO:8) L2 c-Jun TGA ATA CAT TTA TTG TG*A* C (SEQ ID NO:9) L1 RightPrimers (5′ to 3′) c-Jun CGA CTG TAG GAG GGC AG*C* G (SEQ ID NO:10) R1c-Jun CGT CAG CCC ACA ATG CA*C* C (SEQ ID NO:11) R2 c-Jun GTA CTT GGATTC TCA GC*C* T (SEQ ID NO:12) R3 c-Jun CAA ATC TCT CGG CTT CT*A* C (SEQID NO:13) R4 c-Jun CGT GTT GTG TTA AGC GT*G* T (SEQ ID NO:14) R5 c-JunCCG CGG AAA AGG AAC CA*C* T (SEQ ID NO:15) R6 c-Jun CTC CTG GCA GCC CAGTG*A* G (SEQ ID NO:16) R7 c-Jun CTC CTC CCC TCG ATG CT*T* C (SEQ IDNO:17) R8 c-Jun CAG TTA CCC TCT GCA GA*T* C (SEQ ID NO:18) R9 c-Jun CTATTT CCT CTG CAG AT*A* A (SEQ ID NO:19) R10

Four reactions were carried out under the following conditions. Humangenomic DNA (50 ng) was placed into a 0.2 ml tube in a total volume of50 μl, yielding final concentrations of 25 mM Tris-HCl, pH 7.5, 50 mMKCl, 10 mM MgCl₂, and 100 μM exonuclease-resistant hexamer or 1 μM eachof exonuclease-resistant nested primers. A heat-treatment step toincrease primer annealing was included or omitted, as indicated, forindividual reactions. Annealing reactions were heated to 95° C. for 3minutes and slowly cooled down to 37° C. in a PCR System Thermocycler(Perkin Elmer). Reactions were divided into two, each had 25 μl and thenwas brought to a final volume of 50 μl, containing final concentrationsof 37 mM Tris-HCl, pH 8.0, 50 mM KCl, 10 mM MgCl₂, 5 mM (NH₄)₂SO₄, 1.0mM dNTPs, 1 unit/ml of yeast pyrophosphatase, 50 μMexonuclease-resistant hexamer or 0.5 μM exonuclease-resistant nestedprimers, and 800 units/ml φ29 DNA polymerase. Radioactively labeledα-[³²P] dCTP, approximately 60 cpm/pmol total dNTPs, was added to one oftwo parallel reactions for quantification of DNA synthesis. Reactionswere incubated for 18 hours at 37° C. Incorporation of acid-precipitableradioactive deoxyribonucleotide product was determined with glass fiberfilters. The reactions that did not contain α-[³²P] dCTP were analyzedby TaqMan® quantitative PCR analysis as described in Example 5. Astandard curve for input template was generated to determine the locuscopy number of the amplified DNA sample relative to that of genomic DNA.The standard curve was generated from 0, 0.001, 0.01, 0.1, 0.5, and 1 μgof genomic DNA. The results are shown in FIG. 7.

The Y-axis is the locus representation relative to input genomic DNA. Itis calculated as the yield of quantitative PCR product from 1 μgamplified DNA divided by the yield from 1 μg genomic DNA control,expressed as a percent.

As can be seen, the representation of c-jun sequences amplified withrandom hexamers from DNA heated to 95° C. was 69% (see RH (heat) bar).The representation of c-jun sequences amplified with nested primers fromDNA heated to 95° C. was only 3% (see NP (heat) bar). The representationof c-jun sequences in DNA amplified with random hexamers withouttemplate DNA heat treatment was 211% (see RH (no heat) bar). Therepresentation of c-jun sequences in DNA amplified with nested primerswithout template DNA heat treatment was 2828% (see NP (no heat) bar).

H. Example 8 Amplification Bias of Loci Amplified by MDA from WholeBlood

This example demonstrates that genomic DNA can be amplified using MDAdirectly from whole blood or from tissue culture cells and that thelocus representation is substantially the same as for DNA amplified frompurified genomic DNA template. This example illustrates lysis bysubjecting cells to alkaline conditions (by addition of a lysissolution) without any lysis by heating, stabilization of the cell lysate(by addition of a stabilization solution), and multiple displacementamplification of genomic DNA in the stabilized cell lysate. Thestabilized cell lysate is used for amplification without purification ofthe genomic DNA. As a control, DNA amplified in the absence of addedtemplate was tested and contains no detectable sequence representationfor these loci.

DNA was prepared from blood or a tissue culture cell line as follows.Human blood samples were obtained from Grove Hill Laboratory. U266, amyeloma cell line, was obtained from ATCC and passaged according to theaccompanying protocol. Cells were lysed in an alkaline lysis solution bya modification of Zhang et al. (Zhang, L. et al. Whole genomeamplification from a single cell: implications for genetic analysis.Proc Natl Acad Sci USA. 89, 5847–5851 (1992)). Blood was diluted 3-foldin PBS (137 mM NaCl, 2.7 mM KCl, 9.5 mM Na, KPO₄, pH 7.4), while tissueculture cells were diluted to 30,000 cells/ml in PBS. Blood or cellswere lysed by dilution with an equal volume of Alkaline Lysis Buffer(400 mM KOH, 100 mM dithiothreitol, and 10 mM EDTA) and incubated 10 minon ice. The lysed cells were neutralized with the same volume ofNeutralization Buffer (400 mM HCl, 600 mM Tris-HCl, pH 7.5).Preparations of lysed blood or cells (1 μl) were used directly astemplate in MDA reactions (100 μl) as described.

MDA reactions (100 μl) were carried out without denaturation at 95° C.as described in Example 5. Reactions using purified human genomic DNAtemplate contained 300 or 30 ng DNA, resulting in approximately 100- or1000-fold DNA amplification. Reactions using DNA from lysed blood orcells contained 1 μl of the stabilized cell lysates as template. Controlamplification reactions contained no added template DNA.

TaqMan® quantitative PCR analysis was performed on amplified DNA samplesas described in Example 5, and the results are shown in FIG. 8. Therelative representation of eight loci for DNA from five differentamplification reactions is depicted in FIG. 8. The Y-axis is the locusrepresentation, expressed as a percent, relative to input genomic DNA,which is calculated as the yield of quantitative PCR product from 1 μgof amplified DNA divided by the yield from 1 μg of genomic DNA control.Bars with declining diagonals depict the locus representation for DNAamplified from whole blood. Solid gray bars depict the locusrepresentation for DNA amplified from 30 ng purified genomic DNA (9,000genome copies). Solid white bars depict the locus representation for DNAamplified from 300 ng purified genomic DNA (90,000 genome copies). Barswith rising diagonals depict the locus representation for DNA amplifiedfrom tissue culture cells (10 cell equivalents of DNA). Solid black barsdepict the locus representation for DNA amplified from reactions with noadded template (the values for the data represented by the black barsare so small that the bars are not visible on the graph).

As can be seen, DNA amplified directly from whole blood or from tissueculture cells without purification has substantially the same values forlocus representation as DNA amplified from purified genomic DNAtemplate.

I. Example 9 The Amplification Bias of Loci Amplified by MDA inReactions Containing AAdUTP is the Same as the Amplification Bias of DNAAmplified in Reactions Containing 100% dTTP

This example demonstrates that genomic DNA can be amplified using MDA inreactions containing AAdUTP (5-(3-aminoallyl)-2′-deoxyuridine5′-triphosphate, Sigma-Aldrich Co.) and that the locus representation issubstantially the same as for DNA amplified in reactions containing onlydTTP.

MDA reactions (100 μl) containing 100% dTTP were carried out withoutdenaturation at 95° C. as described in Example 5. Reactions containing70% AAdUTP were carried out under the same conditions as reactionscontaining 100% dTTP, with the exception that they contained 0.7 mMAAdUTP and 0.3 mM dTTP, instead of the standard 1.0 mM dTTP. Reactionscontained 1 ng human genomic DNA template, resulting in approximately30,000-fold DNA amplification.

TaqMan® quantitative PCR analysis was performed on amplified DNA samplesas described in Example 5, and the relative representation of eight locifor DNA from two amplification reactions is depicted in FIG. 9. TheY-axis is the locus representation, expressed as a percent, relative toinput genomic DNA, which is calculated as the yield of quantitative PCRproduct from 1 μg of amplified DNA divided by the yield from 1 μg ofgenomic DNA control. Black bars depict the locus representation for DNAamplified in a reaction containing 100% dTTP. White bars depict thelocus representation for DNA amplified in a reaction containing 30%dTTP/70% AAdUTP.

As can be seen, DNA amplified in reactions containing 30% dTTP/70%AAdUTP has substantially the same values for locus representation as DNAamplified in reactions containing 100% dTTP.

J. Example 10 Amplification of c-jun Sequences from Human Genomic DNAusing Sequence Specific Primers and Target Circularization

This example describes an embodiment of the disclosed method andanalysis of the resulting DNA products. The exemplified method is thedisclosed gene-specific multiple displacement amplification form ofmultiple strand displacement amplification using nuclease-resistantsequence-specific primers and circularized DNA template.

Amplification reactions were carried out without a heatdenaturing/annealing step under the conditions described in Example 5using exonuclease-resistant sequence-specific primers that hybridize tosequences within a 5.5 kb EcoRI fragment that contains the c-junsequence. The sequence-specific primers used in this example are listedin Table 3. An asterisk in the nucleotide sequence indicates thepresence of a phosphorothioate bond. The sequence-specific primers aredesigned to hybridize to opposite strands on each side of the c-jun genesequence, and the primers encompass a 2025 bp fragment containing thec-jun gene sequence. The primers are spaced 150–400 nucleotides betweeneach other on each side of the c-jun gene sequence. The region of humanDNA encompassing the recognition sites for these primers can be accessedfrom Genbank using Accession Number AL136985 and spans positions 66962to 68987 of the nucleotide sequence.

TABLE 3 Left Primers (5′ to 3′) c-Jun CTG AAA CAT CGC ACT AT*C *C (SEQID NO:21) D1 c-Jun CCA AAC TTT GAA ATG TT*T *G (SEQ ID NO:22) D2 c-JunCTG CCA CCA ATT CCT GC*T *T (SEQ ID NO:23) D3 c-Jun CAT AAG CAA AGG CCATC*T *T (SEQ ID NO:24) D4 c-Jun GGA AGC AAT TCA AGA TC*T *G (SEQ IDNO:25) D5 c-Jun CTT CAG ATT GCA GCA AT*G *T (SEQ ID NO:26) D6 c-Jun GAATTA ATG AAA TTG GG*A *G (SEQ ID NO:27) D7 c-Jun ACT GTT AGT TTC CAT AG*G*C (SEQ ID NO:28) D8 c-Jun CAA GGT TGA TTA TTT TA*G *A (SEQ ID NO:29) D9c-Jun AGT ACT AGT TCA TGT TT*T *C (SEQ ID NO:30) D10 Right Primers (5′to 3′) c-Jun TAG TAC TCC TTA AGA AC*A *C (SEQ ID NO:31) U1 c-Jun CTA ACATTC GAT CTC AT*T *C (SEQ ID NO:32) U2 c-Jun GCG GAC GGG CTG TCC CC*G *C(SEQ ID NO:33) U3 c-Jun GGA AGG ACT TGG CGC GC*C *C (SEQ ID NO:34) U4c-Jun AAC TAA AGC CAA GGG TA*T *C (SEQ ID NO:35) U5 c-Jun ATA ACA CAGAGA GAC A*G *A (SEQ ID NO:36) U6 c-Jun CAA CTC ATG CTA ACG CA*G *C (SEQID NO:37) U7 c-Jun GGA AGC TGG AGA GAA TC*G *C (SEQ ID NO:38) U8 c-JunGAC ATG GAG TCC CAG GA*G *C (SEQ ID NO:39) U9 c-Jun AGG CCC TGA AGG AGGAG*C *C (SEQ ID NO:40) U10

Four reactions were carried out under the following conditions. Humangenomic DNA (5 μg, Coriell Cell Repositories) was digested with 100units of EcoRI for 3 hours in 50 μl according to the manufacturersconditions. The EcoRI endonuclease was inactivated by incubation at 65°C. for 30 min. Digested DNA fragments (0.5 μg) were circularized in an840 μl volume using 1.7 units (Weiss units) of T4 DNA ligase. Thereaction was incubated for 16 h at 4° C. A mock ligation was carried outunder identical conditions except for the omission of DNA ligase. Twoamplification reactions utilizing a portion of the ligated mixture astemplate (16.8 μl; 10 ng DNA) were carried out, one withsequence-specific primers at a concentration of 2.5 μM each and theother with the primers at a concentration of 0.5 μM each. Two moreamplification reactions were carried out utilizing the mock-ligated DNAas template with sequence-specific primer concentrations of 2.5 μM and0.5 μM. Radioactively labeled α-[³²P] dCTP, approximately 60 cpm/pmoltotal dNTPs, was added to parallel reactions for quantification of DNAsynthesis. Reactions were incubated for 18 hours at 37° C. The reactionsthat did not contain α-[³²P] dCTP were analyzed by TaqMan® quantitativePCR analysis as described in Example 5. A standard curve for inputtemplate was generated to determine the locus copy number of theamplified DNA sample relative to that of genomic DNA. The standard curvewas generated from 0, 0.001, 0.01, 0.1, 0.5, and 1 μg of genomic DNA.The results are shown in FIG. 10. The Y-axis is the locus representationrelative to input genomic DNA. It is calculated as the yield ofquantitative PCR product from 1 μg amplified DNA divided by the yieldfrom 1 μg genomic DNA control, expressed as a percent.

As can be seen, the representation of c-jun sequences amplified with 2.5μM Gene-Specific primers from ligated DNA was below detection (see GS2.5+L bar). The representation of c-jun sequences amplified with 2.5 μMGene-Specific primers from mock-ligated DNA was also not detected (seeGS 2.5−L bar). The representation of c-jun sequences in DNA amplifiedwith 0.5 μM Gene-Specific primers from ligated DNA was 32000% (see GS0.5+L bar). The representation of c-jun sequences in DNA amplified with0.5 μM Gene-Specific primers from mock-ligated DNA was also not detected(see GS 0.5−L bar). These results demonstrate a 320-fold amplificationof the c-jun sequences using gene-specific primers and circularizedtemplate DNA. Digested DNA that was not ligated did not show anyappreciable amplification. Only ligated DNA that was amplified with 0.5μM primers was amplified, while 2.5 μM primers did not yield anyamplification.

The specificity of the DNA amplification reaction carried out with 2.5μM Gene-Specific primers and circularized DNA template was tested bycomparing it to the amount of DNA amplification observed at seven otherloci. TaqMan® quantitative PCR analysis was performed as described inExample 5, and the results are shown in FIG. 11. The relativerepresentation of the c-jun locus and seven other loci is depicted andthe seven loci showed only low levels of amplification, indicating thatthe c-jun sequences were specifically amplified using the c-jun specificprimers.

K. Example 11 Increasing the Quality of Multiple DisplacementAmplification of Degraded Genomic DNA by Repairing the Damaged Sample

This example demonstrates that the quality of amplified DNA in WGA isincreased when the damaged genomic DNA is pretreated with a repairmethod by denatured with 70° C. heat in the presence of 15 mM NaOH,adding Tris-HCl pH 4.0 and original sample back to the denatured sample,then slow cooling to hybridize the damaged DNA. The damaged gDNA wascreated by sonicating intact genomic DNA to an average length of 1 kb.This degraded sample was then pretreated with the repair method andplaced into a multiple displacement amplification reaction. The qualityof the amplification products were assessed by a Taqman assay whichcompares the representation of a particular locus on a chromosome of 1μg of amplification product to a 1 μg standard genomic DNA that has notbeen amplified. This example demonstrates that damaged genomic DNA withthe repair treatment has 3-fold improvement in locus representation ofthe amplified products compared to amplified products of damaged genomicDNA without the repair method.

The damaged genomic DNA was created as follows: Promega's genomic DNAsample was diluted to 100 ngs/μl in dH₂O. The sample was sonicated witha 550 Sonic Demembrator by Fisher Scientific setting 40% for 9 seconds.The sample was diluted further to 5 ng/μl and 25 ngs total was used todenature with 15 mM NaOH final and heated to 70° C. for 5 minutes. At70° C., Tris-HCl pH 4.0 was added to create a final pH of 8.0 and 25 ngof native original denatured samples was also added. Amplification bymultiple displacement synthesis was performed on repaired damaged DNAand damaged DNA, and a Control intact DNA. The results in FIG. 12 showthat the repair of the damaged DNA has significantly improved theamplification of the damaged sample compared to amplification of damagedDNA without repair.

L. Example 12 Increasing the Quality of Multiple DisplacementAmplification of Real Genomic DNA Samples Damaged by Prolonged Storageby Repairing the Damaged Sample

This example demonstrates that the quality of amplified product bymultiple displacement amplification of genomic DNA samples damaged byprolong storage was improved by the repair method.

The damaged genomic DNA samples were pretreated with a repair method bydenaturing with 70° C. heat in the presence of 15 mM NaOH, addingTris-HCl pH 4.0 and original sample back to the denatured sample, slowcooling to hybridize the damaged DNA, then placing into a multipledisplacement amplification reaction. The quality of the amplificationproducts were assessed by a Taqman assay as described in Example 11.

As seen in FIG. 13, the quality of the amplification products of thedamaged genomic DNA samples once repaired is significantly improved. Onaverage, a 3-fold improvement in the quality of the amplificationproducts was observed.

M. Example 13 Optimization of the Denaturation Step: Ionic Strength,Heat and NaOH is Results in Improved Repair

This example demonstrates that ionic strength, heat and NaOH are usefulfor the denaturation step in the disclosed method of repairing andamplifying damaged DNA. Damaged DNA samples were prepared as follows:DNA samples was diluted in dH₂O to 5 ng/μl and were heated to 70° C. for5 minutes which degrades the samples into smaller fragments. The heatedDNA samples were slow cooled and multiple displacement amplification wasperformed on these samples. A Taqman analysis of the amplificationproducts from the heated DNA substrate showed that use of an ionicsolution led to further degradation of the DNA, ultimately leading topoor amplification of these samples.

To determine if heating the damaged DNA in the presence of salt canimprove the amplification of this substrate, damaged DNA (25 ngs) washeated to 70° C. in the presence of a salt (Tris-EDTA (10 mM Tris pH 8.0and 1 mM EDTA)) for 3 minutes, 25 ngs of native damaged sample was addedback, and then the sample was cooled. This method showed someimprovement on the repair of the damaged DNA. When 25 mM final of NaOHwas added to the reaction before the heat step, this improved the repairmethod giving consistent repair of the damaged DNA. This was done bydiluting damaged DNA in Tris-EDTA (TE) to 5 ng/μl, 25 ngs of DNA wasadded to NaOH final concentration 25 mM, the sample was heated to 70° C.for 3 minutes, Tris-HCl pH 4.0 was added to reduce the pH to 8.0, 25 ngsof the original native damaged DNA sample was added back, and themixture was cooled to room temperature. Repair with heat, Tris, and NaOHgave better locus representation than the no repair and repair with heatand Tris only treatments.

N. Example 14 Neutralization Step of the Repair Method ImprovesAmplification of Damaged DNA Sample

This example demonstrates that the neutralization of the repair methodis important for improved quality of amplification products. A damagedDNA sample was prepared as follows: a DNA sample was diluted in dH₂O to10 ng/μl and was heated to 70° C. for 5 minutes to create damaged DNAsamples. The heated DNA sample was diluted to 5 ngs/μl in TE, 25 ngs ofdamaged DNA was added to final 25 mM NaOH, heated to 70° C. for 3minutes and was either neutralized with Tris-HCl pH 4.0 to a pH of 8.0and slow cooled or was slow cooled without neutralization. Amplificationreactions of these samples were performed and the quality of productswere analyzed by Taqman analysis. Repair with neutralization gave abetter locus representation than the no repair and repair withoutneutralization treatments.

It is understood that the disclosed invention is not limited to theparticular methodology, protocols, and reagents described as these mayvary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention which will belimited only by the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a ”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to “ahost cell” includes a plurality of such host cells, reference to “theantibody” is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices, and materials are as described. Publications cited herein andthe material for which they are cited are specifically incorporated byreference. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method of amplifying a whole genome, the method comprising, exposing cells to alkaline conditions to form a cell lysate, wherein the cell lysate comprises a whole genome, reducing the pH of the cell lysate to form a stabilized cell lysate, and incubating the stabilized cell lysate under conditions that promote replication of the genome, wherein replication of the genome results in replicated strands, wherein during replication at least one of the replicated strands is displaced from the genome by strand displacement replication of another replicated strand.
 2. The method of claim 1 wherein the cells are exposed to alkaline conditions by mixing the cells with a lysis solution.
 3. The method of claim 2 wherein the lysis solution comprises a base.
 4. The method of claim 3 wherein the base is an aqueous base.
 5. The method of claim 3 wherein the base is potassium hydroxide, sodium hydroxide, potassium acetate, sodium acetate, ammonium hydroxide, lithium hydroxide, calcium hydroxide, magnesium hydroxide, sodium carbonate, sodium bicarbonate, calcium carbonate, ammonia, aniline, benzylamine, n-butylamine, diethylamine, dimethylamine, diphenylamine, ethylamine, ethylenediamine, methylamine, N-methylaniline, morpholine, pyridine, triethylamine, trimethylamine, aluminum hydroxide, rubidium hydroxide, cesium hydroxide, strontium hydroxide, barium hydroxide, or DBU (1,8-diazobicyclo[5,4,0]undec-7-ene).
 6. The method of claim 5 wherein the base is potassium hydroxide.
 7. The method of claim 6 wherein the lysis solution comprises 400 mM KOH.
 8. The method of claim 7 wherein the lysis solution comprises 400 mM KOH, 100 mM dithiothreitol, and 10 mM EDTA.
 9. The method of claim 8 wherein the lysis solution consists of 400 mM KOH, 100 mM dithiothreitol, and 10 mM EDTA.
 10. The method of claim 3 wherein the lysis solution comprises a plurality of basic agents.
 11. The method of claim 2 wherein the cells are mixed with an equal volume of the lysis solution.
 12. The method of claim 11 wherein the lysis solution comprises 400 mM KOH, 100 mM dithiothreitol, and 10 mM EDTA.
 13. The method of claim 2 wherein the lysis solution comprises a buffer.
 14. The method of claim 13 wherein the buffer is a phosphate buffer, Good buffer, BES, BICINE, CAPS, EPPS, HEPES, MES, MOPS, PIPES, TAPS, TES, TRICINE, sodium cacodylate, sodium citrate, triethylammonium acetate, triethylammonium bicarbonate, Tris, Bis-tris, or Bis-tris propane.
 15. The method of claim 13 wherein the lysis solution comprises a plurality of buffering agents.
 16. The method of claim 1 wherein the pH of the cell lysate is reduced to the range of about pH 7.0 to about pH 6.8.
 17. The method of claim 1 wherein the pH of the cell lysate is reduced by mixing the cell lysate with a stabilization solution.
 18. The method of claim 17 wherein the stabilization solution comprises a buffer.
 19. The method of claim 18 wherein the buffer is phosphate buffer, Good buffer, BES, BICINE, CAPS, EPPS, HEPES, MES, MOPS, PIPES, TAPS, TES, TRICINE, sodium cacodylate, sodium citrate, triethylammonium acetate, triethylammonium bicarbonate, Tris, Bis-tris, or Bis-tris propane.
 20. The method of claim 19 wherein the buffer is Tris-HCl at pH 4.1.
 21. The method of claim 20 wherein the stabilization solution comprises 800 mM Tris-HCl, pH 4.1.
 22. The method of claim 21 wherein the stabilization solution consists of 800 mM Tris-HCl, pH 4.1.
 23. The method of claim 18 wherein the stabilization solution comprises a plurality of buffering agents.
 24. The method of claim 17 wherein the cell lysate is mixed with an equal volume of the stabilization solution.
 25. The method claim 24 wherein the stabilization solution comprises 800 mM Tris-HCl, pH 4.1.
 26. The method of claim 17 wherein the stabilization solution comprises an acid.
 27. The method of claim 26 wherein the acid is hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, acetylsalicylic acid, ascorbic acid, carbonic acid, citric acid, formic acid, nitric acid, perchloric acid, HF, HBr, HI, H₂S, HCN, HSCN, HClO, monochloroacetic acid, dichloroacetic acid, trichloroacetic acid, or a carboxylic acid.
 28. The method of claim 27 wherein the carboxylic acid is ethanoic, propanoic, or butanoic.
 29. The method of claim 17 wherein the stabilization solution comprises a plurality of acidic agents.
 30. The method of claim 1 wherein the pH of the cell lysate is reduced to about pH 9.0 or below.
 31. The method of claim 1 wherein the pH of the cell lysate is reduced to about pH 8.5 or below.
 32. The method of claim 1 wherein the pH of the cell lysate is reduced to about pH 8.0 or below.
 33. The method of claim 1 wherein the pH of the cell lysate is reduced to about pH 7.5 or below.
 34. The method of claim 1 wherein the pH of the cell lysate is reduced to the range of about pH 9.0 to about pH 6.0.
 35. The method of claim 1 wherein the pH of the cell lysate is reduced to the range of about pH 9.0 to about pH 7.0.
 36. The method of claim 1 wherein the pH of the cell lysate is reduced to the range of about pH 9.0 to about pH 7.5.
 37. The method of claim 1 wherein the pH of the cell lysate is reduced to the range of about pH 9.0 to about pH 8.0.
 38. The method of claim 1 wherein the pH of the cell lysate is reduced to the range of about pH 8.5 to about pH 6.0.
 39. The method of claim 1 wherein the pH of the cell lysate is reduced to the range of about pH 8.5 to about pH 7.0.
 40. The method of claim 1 wherein the pH of the cell lysate is reduced to the range of about pH 8.5 to about pH 7.5.
 41. The method of claim 1 wherein the pH of the cell lysate is reduced to the range of about pH 8.5 to about pH 8.0.
 42. The method of claim 1 wherein the pH of the cell lysate is reduced to the range of about pH 8.0 to about pH 6.0.
 43. The method of claim 1 wherein the pH of the cell lysate is reduced to the range of about pH 8.0 to about pH 6.5.
 44. The method of claim 1 wherein the pH of the cell lysate is reduced to the range of about pH 8.0 to about pH 7.0.
 45. The method of claim 1 wherein the pH of the cell lysate is reduced to the range of about pH 8.0 to about pH 7.5.
 46. The method of claim 1 wherein the pH of the cell lysate is reduced to the range of about pH 7.5 to about pH 6.0.
 47. The method of claim 1 wherein the pH of the cell lysate is reduced to the range of about pH 7.5 to about pH 7.0.
 48. The method of claim 1 wherein nucleic acids in the cell lysate and the stabilized cell lysate are not separated from other material in the cell lysate.
 49. The method of claim 1 wherein the cell lysate and the stabilized cell lysate are not subjected to purification prior to the incubation.
 50. The method of claim 49 wherein the purification comprises separation of nucleic acids in the cell lysate from other material in the cell lysate.
 51. The method of claim 49 wherein the purification comprises centrifugation, extraction, chromatography, filtration, dialysis, or a combination of these.
 52. The method of claim 49 wherein the purification comprises precipitation other than precipitation caused by the alkaline conditions or by the reduction of the pH.
 53. The method of claim 49 wherein the purification comprises centrifugation, phenol-chloroform extraction, column chromatography, or a combination of these.
 54. The method of claim 1 wherein the cell lysate, stabilized cell lysate, or both are subjected to partial purification prior to the incubation.
 55. The method of claim 54 wherein the partial purification comprises centrifugation, extraction, chromatography, precipitation, filtration, dialysis, or a combination of these.
 56. The method of claim 54 wherein the partial purification comprises centrifugation, phenol-chloroform extraction, column chromatography, or a combination of these.
 57. The method of claim 1 wherein the cell lysate and the stabilized cell lysate are not subjected to substantial purification prior to the incubation.
 58. The method of claim 57 wherein the substantial purification does not include centrifugation, extraction, chromatography, precipitation, filtration, or dialysis.
 59. The method of claim 57 wherein the substantial purification does not include centrifugation, phenol-chloroform extraction, or column chromatography.
 60. The method of claim 57 wherein the cell lysate, stabilized cell lysate, or both are subjected to centrifugation, extraction, chromatography, precipitation, filtration, or dialysis prior to the incubation.
 61. The method of claim 57 wherein the cell lysate, stabilized cell lysate, or both are subjected to centrifugation, phenol-chloroform extraction, or column chromatography prior to the incubation.
 62. The method of claim 57 wherein the substantial purification comprises centrifugation, extraction, chromatography, filtration, dialysis, or a combination of these.
 63. The method of claim 57 wherein the substantial purification comprises precipitation other than precipitation caused by the alkaline conditions or by the reduction of the pH.
 64. The method of claim 57 wherein the substantial purification comprises centrifugation, phenol-chloroform extraction, column chromatography, or a combination of these.
 65. The method of claim 1 wherein the cell lysate and the stabilized cell lysate are not purified prior to the incubation.
 66. The method of claim 1 wherein the cell lysate, stabilized cell lysate, or both are partially purified prior to the incubation.
 67. The method of claim 1 wherein the incubation is substantially isothermic.
 68. The method of claim 67 wherein neither the cell lysate nor the stabilized cell lysate is heated substantially above the temperature of the incubation.
 69. The method of claim 67 wherein neither the cell lysate nor the stabilized cell lysate is subjected to substantial heating above the temperature of the incubation.
 70. The method of claim 67 wherein the cells are not heated substantially above the temperature of the incubation.
 71. The method of claim 67 wherein the cells are not subjected to substantial heating above the temperature of the incubation.
 72. The method of claim 67 wherein the cells are not heated substantially above the temperature at which the cells grow.
 73. The method of claim 67 wherein the cells are not subjected to substantial heating above the temperature at which the cells grow.
 74. The method of claim 67 wherein the cell lysate, stabilized cell lysate, and the cells are not heated substantially above the temperature of the incubation.
 75. The method of claim 67 wherein the cell lysate, stabilized cell lysate, and the cells are not subjected to substantial heating above the temperature of the incubation.
 76. The method of claim 67 wherein the cell lysate, stabilized cell lysate, and the cells are not heated, prior to or during the incubation, substantially above the temperature at which the cells grow.
 77. The method of claim 67 wherein the cell lysate, stabilized cell lysate, and the cells are not subjected to, prior to or during the incubation, substantial heating above the temperature at which the cells grow prior.
 78. The method of claim 1 wherein neither the cell lysate nor the stabilized cell lysate is heated above a temperature and for a time that would cause notable denaturation of the genome.
 79. The method of claim 1 wherein neither the cell lysate nor the stabilized cell lysate is subjected to heating above a temperature and for a time that would cause notable denaturation of the genome.
 80. The method of claim 1 wherein the cells are not lysed by heat.
 81. The method of claim 1 wherein the cells are not heated above a temperature and for a time that would cause substantial cell lysis in the absence of the alkaline conditions.
 82. The method of claim 1 wherein the cells are not subjected to heating above a temperature and for a time that would cause substantial cell lysis in the absence of the alkaline conditions.
 83. A method of amplifying a whole genome, the method comprising, exposing cells to alkaline conditions to form a cell lysate, wherein the cell lysate comprises a whole genome, wherein the cells are exposed to alkaline conditions by mixing the cells with a lysis solution, reducing the pH of the cell lysate to form a stabilized cell lysate, wherein the pH of the cell lysate is reduced by mixing the cell lysate with a stabilization solution, and incubating the stabilized cell lysate under conditions that promote replication of the genome, wherein replication of the genome results in replicated strands, wherein during replication at least one of the replicated strands is displaced from the genome by strand displacement replication of another replicated strand.
 84. The method of claim 83 wherein the lysis solution comprises potassium hydroxide.
 85. The method of claim 84 wherein the lysis solution comprises 400 mM KOH.
 86. The method of claim 85 wherein the lysis solution comprises 400 mM KOH, 100 mM dithiothreitol, and 10 mM EDTA.
 87. The method of claim 86 wherein the lysis solution consists of 400 mM KOH, 100 mM dithiothreitol, and 10 mM EDTA.
 88. The method of claim 83 wherein the cells are mixed with an equal volume of the lysis solution.
 89. The method of claim 88 wherein the lysis solution comprises 400 mM KOH, 100 mM dithiothreitol, and 10 mM EDTA.
 90. The method of claim 83 wherein the stabilization solution comprises Tris-HCl at pH 4.1.
 91. The method of claim 90 wherein the stabilization solution comprises 800 mM Tris-HCl, pH 4.1.
 92. The method of claim 91 wherein the stabilization solution consists of 800 mM Tris-HCl, pH 4.1.
 93. The method of claim 83 wherein the cell lysate is mixed with an equal volume of the stabilization solution.
 94. The method claim 93 wherein the stabilization solution comprises 800 mM Tris-HCl, pH 4.1.
 95. The method of claim 83 wherein the lysis solution consists of 400 mM KOH and 10 mM EDTA, wherein the stabilization solution consists of 800 mM Tris-HCl, pH 4, wherein the stabilized cell lysate is incubated in the presence of 37.5 mM Tris-HCl, 50 mM KCl, 10 mM MgCl₂, 5 mM (NH₄)₂SO₄, 1 mM deoxynucleotide triphosphates, 50 μM primers, and φ29 DNA Polymerase.
 96. The method of claim 95 wherein the stabilized cell lysate is incubated in the presence of 37.5 mM Tris-HCl, 50 mM KCl, 10 mM MgCl₂, 5 mM (NH₄)₂SO₄, 1 mM deoxynucleotide triphosphates, 50 μM primers, and φ29 DNA Polymerase by mixing the stabilized cell lysate with one quarter volume of reaction mix, and φ29 DNA Polymerase, wherein the reaction mix consists of 150 mM Tris-HCl, 200 mM KCl, 40 mM MgCl₂, 20 mM (NH₄)₂SO₄, 4 mM deoxynucleotide triphosphates, and 0.2 mM primers.
 97. A method of amplifying a whole genome, the method comprising, exposing cells to alkaline conditions to form a cell lysate, wherein the cell lysate comprises a whole genome, wherein the cells are exposed to alkaline conditions by mixing the cells with a lysis solution, wherein the lysis solution comprises 400 mM KOH, 100 mM dithiothreitol, and 10 mM EDTA, reducing the pH of the cell lysate to form a stabilized cell lysate, wherein the pH of the cell lysate is reduced by mixing the cell lysate with a stabilization solution, wherein the stabilization solution comprises 800 mM Tris-HCl, pH 4.1, and incubating the stabilized cell lysate under conditions that promote replication of the genome, wherein replication of the genome results in replicated strands, wherein during replication at least one of the replicated strands is displaced from the genome by strand displacement replication of another replicated strand.
 98. The method of claim 97 wherein the cells are mixed with an equal volume of the lysis solution.
 99. The method of claim 97 wherein the cell lysate is mixed with an equal volume of the stabilization solution.
 100. A method of amplifying damaged DNA, the method comprising exposing a damaged DNA sample to conditions that promote substantial denaturation of damaged DNA in the damaged DNA sample, thereby forming a denatured damaged DNA sample, altering the conditions to conditions that do not promote substantial denaturation of damaged DNA in the damaged DNA sample to form a stabilized damaged DNA sample, incubating damaged DNA in the stabilized damaged DNA sample under conditions that promote replication of the damaged DNA, wherein replication of the damaged DNA results in a longer average fragment length for the replicated damaged DNA than the average fragment length in the damaged DNA sample, wherein during replication at least one of the replicated strands is displaced by strand displacement replication of another replicated strand.
 101. The method of claim 100 wherein the damaged DNA sample, the denatured damaged DNA sample, or both are exposed to ionic conditions.
 102. The method of claim 101 wherein the damaged DNA sample and denatured damaged DNA sample are exposed to ionic conditions by mixing an ionic solution with the damaged DNA sample.
 103. The method of claim 102 wherein the ionic solution is mixed with the damaged DNA sample prior to or during exposure of the damaged DNA sample to conditions that promote substantial denaturation of the damaged DNA.
 104. The method of claim 102 wherein the ionic solution is a salt solution.
 105. The method of claim 104 wherein the salt solution comprises one or more salts.
 106. The method of claim 105 wherein the salt is Tris-HCl, Tris-EDTA, sodium chloride, potassium chloride, magnesium chloride, sodium acetate, potassium acetate, magnesium acetate, or a combination.
 107. The method of claim 106 wherein the Tris-HCl is from pH 7.0 to 8.0.
 108. The method of claim 106 wherein the salt is Tris-EDTA.
 109. The method of claim 108 wherein the salt solution comprises about 50 mM to about 500 mM Tris and about 1 mM to about 5 mM EDTA.
 110. The method of claim 109 wherein the ionic solution is diluted 2 to 5 fold when mixed with the damaged DNA sample.
 111. The method of claim 101 wherein the denatured damaged DNA sample is exposed to ionic conditions by mixing an ionic solution with the denatured damaged DNA sample.
 112. The method of claim 111 wherein the ionic solution is mixed with the denatured damaged DNA sample prior to or during altering of the conditions.
 113. The method of claim 100 wherein the damaged DNA sample is exposed to conditions that promote substantial denaturation by mixing the damaged DNA sample with a denaturing solution and by heating the damaged DNA sample to a temperature and for a length of time that substantially denatures the damaged DNA in the damaged DNA sample.
 114. The method of claim 113 wherein the damaged DNA sample is mixed with the denaturing solution after the DNA sample is heated.
 115. The method of claim 113 wherein the damaged DNA sample is mixed with the denaturing solution before the DNA sample is heated.
 116. The method of claim 113 wherein the damaged DNA sample is mixed with the denaturing solution at the same time the DNA sample is heated.
 117. The method of claim 113 wherein the damaged DNA sample is mixed with the denaturing solution during heating of the DNA sample.
 118. The method of claim 113 wherein the damaged DNA sample is mixed with the denaturing solution when heating of the DNA sample begins.
 119. The method of claim 113 wherein mixing the damaged DNA sample with a denaturing solution produces alkaline conditions in the damaged DNA sample.
 120. The method of claim 119 wherein the denaturing solution comprises a base.
 121. The method of claim 120 wherein the base is an aqueous base.
 122. The method of claim 120 wherein the base is sodium hydroxide, potassium hydroxide, potassium acetate, sodium acetate, ammonium hydroxide, lithium hydroxide, calcium hydroxide, magnesium hydroxide, sodium carbonate, sodium bicarbonate, calcium carbonate, ammonia, aniline, benzylamine, n-butylamine, diethylamine, dimethylamine, diphenylamine, ethylamine, ethylenediamine, methylamine, N-methylaniline, morpholine, pyridine, triethylamine, trimethylamine, aluminum hydroxide, rubidium hydroxide, cesium hydroxide, strontium hydroxide, barium hydroxide, or DBU (1,8-diazobicyclo[5,4,0]undec-7-ene).
 123. The method of claim 122 wherein the base is sodium hydroxide.
 124. The method of claim 123 wherein the denaturing solution comprises about 150 mM to about 1 M NaOH.
 125. The method of claim 119 wherein the denaturing solution is 10× concentration, wherein the damaged DNA sample is mixed with the denaturing solution to create a 1× concentration.
 126. The method of claim 119 wherein the alkaline conditions comprise 15 to 50 mM NaOH.
 127. The method of claim 113 wherein the damaged DNA in the damaged DNA sample is substantially denatured without further damaging the DNA.
 128. The method of claim 113 wherein the damaged DNA sample is heated to a temperature of about 70° C. or less and for a length of time of about 5 minutes or less.
 129. The method of claim 113 wherein the temperature is about 60° C. to about 70° C.
 130. The method of claim 113 wherein the temperature is about 50° C. to about 60° C.
 131. The method of claim 113 wherein the temperature is about 40° C. to about 50° C.
 132. The method of claim 113 wherein the temperature is about 25° C. to about 40° C.
 133. The method of claim 113 wherein the temperature is about 60° C. to about 70° C. and the length of time is about 3 minutes.
 134. The method of claim 113 wherein the temperature is about 25° C. to about 50° C. and the length of time is about 5 minutes or more.
 135. The method of 113 wherein altering the conditions comprises reducing the pH of and cooling the denatured damaged DNA sample.
 136. The method of claim 135 wherein the temperature to which the damaged DNA sample is heated is maintained during reduction of the pH of the denatured damaged DNA sample.
 137. The method of claim 135 wherein the temperature to which the damaged DNA sample is heated is reduced before reduction of the pH of the denatured damaged DNA sample.
 138. The method of claim 135 wherein the temperature to which the damaged DNA sample is heated is reduced during reduction of the pH of the denatured damaged DNA sample.
 139. The method of claim 135 wherein cooling the denatured damaged DNA sample is commenced during reduction of the pH of the denatured damaged DNA sample.
 140. The method of claim 135 wherein cooling the denatured damaged DNA sample is commenced when the pH of the denatured damaged DNA sample is reduced.
 141. The method of claim 135 wherein the pH of the denatured damaged DNA sample is reduced by mixing the denatured damaged DNA sample with a stabilization solution.
 142. The method of claim 141 wherein the pH of the denatured damaged DNA sample is reduced to the range of about pH 7.5 to about pH 8.0.
 143. The method of claim 141 wherein the pH of the denatured damaged DNA sample is reduced by mixing the denatured damaged DNA sample with a stabilization solution.
 144. The method of claim 143 wherein the stabilization solution comprises a buffer.
 145. The method of claim 141 wherein the buffer is phosphate buffer, Good buffer, BES, BICINE, CAPS, EPPS, HEPES, MES, MOPS, PIPES, TAPS, TES, TRICINE, sodium cacodylate, sodium citrate, triethylammonium acetate, triethylammonium bicarbonate, Tris, Bis-tris, or Bis-tris propane.
 146. The method of claim 141 wherein the stabilization solution comprises 800 mM Tris-HCl, pH 4.1.
 147. The method of claim 141 wherein the stabilization solution comprises an acid.
 148. The method of claim 147 wherein the acid is hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, acetylsalicylic acid, ascorbic acid, carbonic acid, citric acid, formic acid, nitric acid, perchloric acid, HF, HBr, HI, H₂S, HCN, HSCN, HClO, monochloroacetic acid, dichloroacetic acid, trichloroacetic acid, or a carboxylic acid.
 149. The method of claim 147 wherein the carboxylic acid is ethanoic, propanoic, or butanoic.
 150. The method of claim 147 wherein the stabilization solution comprises a plurality of acidic agents.
 151. The method of claim 141 wherein the pH of the denatured damaged DNA sample is reduced to the range of about pH 9.0 to about pH 6.8.
 152. The method of claim 141 wherein the pH of the denatured damaged DNA sample is reduced to the range of about pH 9.0 to about pH 7.5.
 153. The method of claim 141 wherein the pH of the denatured damaged DNA sample is reduced to the range of about pH 9.0 to about pH 8.0.
 154. The method of claim 141 wherein the pH of the denatured damaged DNA sample is reduced to the range of about pH 8.5 to about pH 6.8.
 155. The method of claim 141 wherein the pH of the denatured damaged DNA sample is reduced to the range of about pH 8.5 to about pH 7.5.
 156. The method of claim 141 wherein the pH of the denatured damaged DNA sample is reduced to the range of about pH 8.5 to about pH 8.0.
 157. The method of claim 141 wherein the pH of the denatured damaged DNA sample is reduced to the range of about pH 8.0 to about pH 6.8.
 158. The method of claim 141 wherein the pH of the denatured damaged DNA sample is reduced to the range of about pH 8.0 to about pH 7.5.
 159. The method of claim 141 wherein the pH of the denatured damaged DNA sample is reduced to about pH 9.0 or less.
 160. The method of claim 141 wherein the pH of the denatured damaged DNA sample is reduced to about pH 8.5 or less.
 161. The method of claim 141 wherein the pH of the denatured damaged DNA sample is reduced to about pH 8.0 or less.
 162. The method of claim 141 wherein the pH of the denatured damaged DNA sample is reduced to about pH 7.5 or less.
 163. The method of claim 141 wherein the stabilization solution comprises one or more salts.
 164. The method of claim 163 wherein the salt is Tris-HCl, Tris-EDTA, sodium chloride, potassium chloride, magnesium chloride, sodium acetate, potassium acetate, magnesium acetate, or a combination.
 165. The method of claim 164 wherein the Tris-HCl is from pH 7.0 to 8.0.
 166. The method of claim 164 wherein the salt is Tris-EDTA.
 167. The method of claim 166 wherein the stabilization solution comprises about 50 mM to about 500 mM Tris and about 1 mM to about 5 mM EDTA.
 168. The method of claim 135 wherein the damaged DNA mixture is cooled at a rate of about 1° C. per minute or less.
 169. The method of claim 135 wherein the damaged DNA mixture is cooled at a rate of about 1% per minute or less.
 170. The method of claim 169 wherein the damaged DNA mixture is cooled to room temperature or lower from 60° C. to 70° C.
 171. The method of claim 169 wherein the damaged DNA mixture is cooled to room temperature or lower from 50 to 60° C.
 172. The method of claim 169 wherein the damaged DNA mixture is cooled to room temperature or lower from 40° C. to 50° C.
 173. The method of claim 169 wherein the damaged DNA mixture is cooled to room temperature or lower from 30° C. to 40° C.
 174. The method of claim 169 wherein the damaged DNA mixture is cooled to room temperature from 50° C. to 70° C.
 175. The method of claim 113 wherein the denaturing solution comprises one or more salts.
 176. The method of claim 175 wherein the salt is Tris-HCI, Tris-EDTA, sodium chloride, potassium chloride, magnesium chloride, sodium acetate, potassium acetate, magnesium acetate, or a combination.
 177. The method of claim 176 wherein the Tris-HCl is from pH 7.0 to 8.0.
 178. The method of claim 176 wherein the salt is Tris-EDTA.
 179. The method of claim 178 wherein the denaturing solution comprises about 50 mM to about 500 mM Tris and about 1 mM to about 5 mM EDTA.
 180. The method of claim 100 wherein the damaged DNA sample is comprised of degraded DNA fragments of genomic DNA.
 181. The method of claim 100 wherein replication and repair of the damaged DNA is accomplished by incubating the damaged DNA in the presence of a DNA polymerase.
 182. The method of claim 181 wherein the polymerase is a DNA polymerase that can extend the 3′-ends of the damaged DNA.
 183. The method of claim 182 wherein the DNA polymerase is φ29 DNA polymerase, BST DNA polymerase, Taq DNA polymerase, a modified form of Taq DNA polymerase, a Reverse Transcriptase, T4 DNA polymerase, T7 DNA polymerase, Pol I DNA polymerase, or a modified form of DNA Polymerase I.
 184. The method of claim 181 wherein the DNA polymerase is φ29 DNA Polymerase.
 185. The method of claim 181 wherein the damaged DNA is amplified using a kit, wherein the kit comprises a denaturing solution, a stabilization solution, a set of primers, and a DNA polymerase.
 186. The method of claim 100 wherein the damaged DNA sample is a cell lysate, wherein the cell lysate is produced by exposing cells to alkaline condition, wherein the cell lysate comprises a whole genome.
 187. The method of claim 1 further comprising following exposure of the cells to alkaline conditions, exposing a first portion of the cell lysate to conditions that promote substantial denaturation of damaged DNA in the first portion of the cell lysate, wherein reducing the pH of the cell lysate comprises reducing the pH of the first portion of the cell lysate to form a first stabilized cell lysate and reducing the pH of a second portion of the cell lysate to form a second stabilized cell lysate, following reducing the pH of the cell lysate, mixing the second stabilized cell lysate with the first stabilized cell lysate under conditions that promote transient denaturation of the ends of damaged DNA in the second stabilized cell lysate and that maintain substantial denaturation of the damaged DNA in the first stabilized cell lysate, thereby forming a stabilized cell lysate mixture, and prior to incubating the stabilized cell lysate, cooling the stabilized cell lysate mixture under conditions that promote annealing of the ends of the transiently denatured damaged DNA to the substantially denatured damaged DNA, wherein incubating the stabilized cell lysate under conditions that promote replication of the genome also promotes replication of the damaged DNA, wherein the annealed ends of the damaged DNA prime replication, wherein replication of the damaged DNA results in repair of the replicated strands.
 188. A method of amplifying damaged DNA, the method comprising exposing a first damaged DNA sample to conditions that promote substantial denaturation of damaged DNA in the first damaged DNA sample, thereby forming a denatured damaged DNA sample, reducing the pH of the denatured damaged DNA sample to form a stabilized denatured damaged DNA sample, mixing a second damaged DNA sample with the stabilized denatured damaged DNA sample under conditions that promote transient denaturation of the ends of damaged DNA in the second sample and that maintain substantial denaturation of the damaged DNA in the stabilized denatured damaged DNA sample, thereby forming a damaged DNA mixture, cooling the damaged DNA mixture under conditions that promote annealing of the ends of the transiently denatured damaged DNA to the substantially denatured damaged DNA, incubating the annealed damaged DNA under conditions that promote replication of the damaged DNA, wherein the annealed ends of the damaged DNA prime replication, wherein replication of the damaged DNA results in repair of the replicated strands, wherein during replication at least one of the replicated strands is displaced by strand displacement replication of another replicated strand.
 189. The method of claim 188 wherein the temperature to which the first damaged DNA sample is heated is maintained during mixing of the second damaged DNA sample with the stabilized denatured damaged DNA sample.
 190. The method of claim 188 wherein the pH of the stabilized denatured damaged DNA sample is not high enough nor low enough to cause further substantial denaturation upon mixing second damaged DNA sample with the stabilized denatured damaged DNA sample.
 191. The method of claim 188 wherein the first damaged DNA sample is a portion of a damaged DNA sample, wherein the second damaged DNA sample is a portion of the same damaged DNA sample.
 192. The method of claim 188 wherein the first damaged DNA sample is from the same source as the second damaged DNA sample.
 193. The method of claim 188 wherein the first damaged DNA sample is from the same organism as the second damaged DNA sample.
 194. The method of claim 188 wherein the first damaged DNA sample is from the same tissue as the second damaged DNA sample.
 195. The method of claim 188 wherein the second damaged DNA sample is mixed with the stabilized denatured damaged DNA sample at a temperature and for a length of time that transiently denatures the damaged DNA in the second damaged DNA sample.
 196. The method of claim 195 wherein the temperature is about 70° C. or less and the length of time is about 30 seconds or less.
 197. The method of claim 195 wherein the second damaged DNA sample is mixed with the stabilized denatured damaged DNA sample at a temperature that does not further damage the DNA.
 198. The method of claim 197 wherein the temperature is about 60° C. to about 70° C.
 199. The method of claim 197 wherein the temperature is about 50° C. to about 60° C.
 200. The method of claim 197 wherein the temperature is about 40° C. to about 50° C.
 201. The method of claim 197 wherein the temperature is about 25° C. to about 40° C.
 202. The method of claim 197 wherein the temperature is about 25° C. to about 70° C. and the length of time is about 30 seconds. 